Modulators of pnpla3 expression

ABSTRACT

The present embodiments provide methods, compounds, and compositions useful for inhibiting PNPLA3 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PNPLA3.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0317USLSEQ_ST25.txt created Sep. 13, 2018, which is 480 kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositionsuseful for inhibiting PNPLA3 (patatin like phospholipase domaincontaining 3; hypothetical protein dJ796I17.1; adiponutrin; DJ796I17.1)expression, and in certain instances, reducing the amount of PNPLA3protein in a cell or animal, which can be useful for treating,preventing, or ameliorating a disease associated with PNPLA3.

BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of liverdisease from steatosis to nonalcoholic steatohepatitis (NASH) andcirrhosis. NAFLD is defined as fat accumulation in the liver exceeding5% by weight, in the absense of significant alcohol consumption,steatogenic medication, or hereditary disorders (Kotronen et al,Arterioscler Thromb. Vasc. Biol. 2008, 28: 27-38).

Non-alcoholic steatohepatitis (NASH) is NAFLD with signs of inflammationand hepatic injury. NASH is defined histologically by macrovesicularsteatosis, hepatocellular ballooning, and lobular inflammatoryinfiltrates (Sanyal, Hepatol. Res. 2011. 41: 670-4). NASH is estimatedto affect 2-3% of the general population. In the presence of otherpathologies, such as obesity or diabetes, the estimated prevalenceincreases to 7% and 62% respectively (Hashimoto et al, J. Gastroenterol.2011. 46(1): 63-69).

PNPLA3 is a 481 amino acid member of the patatin-like phospholipasedomain-containing family that is expressed in the ER and on lipiddroplets. In humans, PNPLA3 is highly expressed in the liver, whereasadipose tissue expression is five-fold less (Huang et al, Proc. Natl.Acad. Sci. USA 2010. 107: 7892-7).

SUMMARY

Certain embodiments provided herein are compounds and methods forreducing the amount or activity of PNPLA3 mRNA, and in certainembodiments, reducing the amount of PNPLA3 protein in a cell or animal.In certain embodiments, the animal has a liver disease. In certainembodiments, the disease is NASH. In certain embodiments, the disease isNAFLD. In certain embodiments, the disease is hepatic steatosis. Incertain embodiments, the disease is liver cirrhosis. In certainembodiments, the disease is hepatocellular carcinoma. In certainembodiments, the disease is alcoholic liver disease. In certainembodiments, the disease is alcoholic steatohepatitis (ASH). In certainembodiments, the disease is HCV hepatitis. In certain embodiments, thedisease is chronic hepatitis. In certain embodiments, the disease ishereditary hemochromatosis. In certain embodiments, the disease isprimary sclerosing cholangitis. Certain compounds provided herein aredirected to compounds and compositions that reduce liver damage,steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation in an animal.

Certain embodiments provided herein are directed to potent and tolerablecompounds and compositions useful for inhibiting PNPLA3 expression,which can be useful for treating, preventing, ameliorating, or slowingprogression of liver diseases. Certain embodiments provided herein aredirected to compounds and compositions that are more potent or havegreater therapeutic value than compounds publicly disclosed.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the embodiments, as claimed. Herein, the useof the singular includes the plural unless specifically statedotherwise. As used herein, the use of “or” means “and/or” unless statedotherwise. Furthermore, the use of the term “including” as well as otherforms, such as “includes” and “included”, is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and GenBank and NCBI reference sequence records arehereby expressly incorporated by reference for the portions of thedocument discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in theexamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, compoundsdefined by a SEQ ID NO may comprise, independently, one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase. Compounds described by ION number indicate a combination ofnucleobase sequence, chemical modification, and motif.

Definitions

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-deoxynucleoside” means a nucleoside comprising 2′-H(H) furanosylsugar moiety, as found in naturally occurring deoxyribonucleic acids(DNA). In certain embodiments, a 2′-deoxynucleoside may comprise amodified nucleobase or may comprise an RNA nucleobase (uracil).

“2′-O-methoxyethyl” (also 2′-MOE) refers to a 2′-O(CH₂)₂—OCH₃) in theplace of the 2′-OH group of a ribosyl ring. A 2′-O-methoxyethyl modifiedsugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means anucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” or “2-modified nucleoside” means anucleoside comprising a 2′-substituted or 2′-modified sugar moiety. Asused herein, “2′-substituted” or “2-modified” in reference to a sugarmoiety means a sugar moiety comprising at least one 2′-substituent groupother than H or OH.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular compound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular compound.

“5-methylcytosine” means a cytosine with a methyl group attached to the5 position.

“About” means within ±10% of a value. For example, if it is stated, “thecompounds affected about 70% inhibition of PNPLA3”, it is implied thatPNPLA3 levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing acompound or composition provided herein to an individual to perform itsintended function. An example of a route of administration that can beused includes, but is not limited to parenteral administration, such assubcutaneous, intravenous, or intramuscular injection or infusion.

“Administered concomitantly” or “co-administration” means administrationof two or more compounds in any manner in which the pharmacologicaleffects of both are manifest in the patient.

Concomitant administration does not require that both compounds beadministered in a single pharmaceutical composition, in the same dosageform, by the same route of administration, or at the same time. Theeffects of both compounds need not manifest themselves at the same time.The effects need only be overlapping for a period of time and need notbe coextensive. Concomitant administration or co-administrationencompasses administration in parallel or sequentially.

“Amelioration” refers to an improvement or lessening of at least oneindicator, sign, or symptom of an associated disease, disorder, orcondition. In certain embodiments, amelioration includes a delay orslowing in the progression or severity of one or more indicators of acondition or disease. The progression or severity of indicators may bedetermined by subjective or objective measures, which are known to thoseskilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable and/or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid compared to target nucleic acid levels ortarget protein levels in the absence of the antisense compound to thetarget.

“Antisense compound” means a compound comprising an oligonucleotide andoptionally one or more additional features, such as a conjugate group orterminal group. Examples of antisense compounds include single-strandedand double-stranded compounds, such as, oligonucleotides, ribozymes,siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels in the absence of theantisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with target nucleic acid, wherein the outcome or effect ofthe hybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

“Antisense oligonucleotide” means an oligonucleotide having a nucleobasesequence that is complementary to a target nucleic acid or region orsegment thereof. In certain embodiments, an antisense oligonucleotide isspecifically hybridizable to a target nucleic acid or region or segmentthereof.

“Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclicsugar moiety. “Bicyclic sugar” or “bicyclic sugar moiety” means amodified sugar moiety comprising two rings, wherein the second ring isformed via a bridge connecting two of the atoms in the first ringthereby forming a bicyclic structure. In certain embodiments, the firstring of the bicyclic sugar moiety is a furanosyl moiety. In certainembodiments, the bicyclic sugar moiety does not comprise a furanosylmoiety.

“Branching group” means a group of atoms having at least 3 positionsthat are capable of forming covalent linkages to at least 3 groups. Incertain embodiments, a branching group provides a plurality of reactivesites for connecting tethered ligands to an oligonucleotide via aconjugate linker and/or a cleavable moiety.

“Cell-targeting moiety” means a conjugate group or portion of aconjugate group that is capable of binding to a particular cell type orparticular cell types.

“cEt” or “constrained ethyl” means a ribosyl bicyclic sugar moietywherein the second ring of the bicyclic sugar is formed via a bridgeconnecting the 4′-carbon and the 2′-carbon, wherein the bridge has theformula: 4′-CH(CH₃)—O-2′, and wherein the methyl group of the bridge isin the S configuration.

“cEt nucleoside” means a nucleoside comprising a cEt modified sugarmoiety.

“Chemical modification” in a compound describes the substitutions orchanges through chemical reaction, of any of the units in the compoundrelative to the original state of such unit. “Modified nucleoside” meansa nucleoside having, independently, a modified sugar moiety and/ormodified nucleobase. “Modified oligonucleotide” means an oligonucleotidecomprising at least one modified internucleoside linkage, a modifiedsugar, and/or a modified nucleobase.

“Chemically distinct region” refers to a region of a compound that is insome way chemically different than another region of the same compound.For example, a region having 2′-O-methoxyethyl nucleotides is chemicallydistinct from a region having nucleotides without 2′-O-methoxyethylmodifications.

“Chimeric antisense compounds” means antisense compounds that have atleast 2 chemically distinct regions, each position having a plurality ofsubunits.

“Cleavable bond” means any chemical bond capable of being split. Incertain embodiments, a cleavable bond is selected from among: an amide,a polyamide, an ester, an ether, one or both esters of a phosphodiester,a phosphate ester, a carbamate, a di-sulfide, or a peptide.

“Cleavable moiety” means a bond or group of atoms that is cleaved underphysiological conditions, for example, inside a cell, an animal, or ahuman.

“Complementary” in reference to an oligonucleotide means the nucleobasesequence of such oligonucleotide or one or more regions thereof matchesthe nucleobase sequence of another oligonucleotide or nucleic acid orone or more regions thereof when the two nucleobase sequences arealigned in opposing directions. Nucleobase matches or complementarynucleobases, as described herein, are limited to the following pairs:adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C)and guanine (G), and 5-methyl cytosine (^(m)C) and guanine (G) unlessotherwise specified. Complementary oligonucleotides and/or nucleic acidsneed not have nucleobase complementarity at each nucleoside and mayinclude one or more nucleobase mismatches. By contrast, “fullycomplementary” or “100% complementary” in reference to oligonucleotidesmeans that such oligonucleotides have nucleobase matches at eachnucleoside without any nucleobase mismatches.

“Conjugate group” means a group of atoms that is attached to anoligonucleotide. Conjugate groups include a conjugate moiety and aconjugate linker that attaches the conjugate moiety to theoligonucleotide.

“Conjugate linker” means a group of atoms comprising at least one bondthat connects a conjugate moiety to an oligonucleotide.

“Conjugate moiety” means a group of atoms that is attached to anoligonucleotide via a conjugate linker.

“Contiguous” in the context of an oligonucleotide refers to nucleosides,nucleobases, sugar moieties, or intermucleoside linkages that areimmediately adjacent to each other. For example, “contiguousnucleobases” means nucleobases that are immediately adjacent to eachother in a sequence.

“Designing” or “Designed to” refer to the process of designing acompound that specifically hybridizes with a selected nucleic acidmolecule.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

“Differently modified” means chemical modifications or chemicalsubstituents that are different from one another, including absence ofmodifications. Thus, for example, a MOE nucleoside and an unmodified DNAnucleoside are “differently modified,” even though the DNA nucleoside isunmodified. Likewise, DNA and RNA are “differently modified,” eventhough both are naturally-occurring unmodified nucleosides. Nucleosidesthat are the same but for comprising different nucleobases are notdifferently modified. For example, a nucleoside comprising a 2′-OMemodified sugar and an unmodified adenine nucleobase and a nucleosidecomprising a 2′-OMe modified sugar and an unmodified thymine nucleobaseare not differently modified.

“Dose” means a specified quantity of a compound or pharmaceutical agentprovided in a single administration, or in a specified time period. Incertain embodiments, a dose may be administered in two or more boluses,tablets, or injections. For example, in certain embodiments, wheresubcutaneous administration is desired, the desired dose may require avolume not easily accommodated by a single injection. In suchembodiments, two or more injections may be used to achieve the desireddose. In certain embodiments, a dose may be administered in two or moreinjections to minimize injection site reaction in an individual. Inother embodiments, the compound or pharmaceutical agent is administeredby infusion over an extended period of time or continuously. Doses maybe stated as the amount of pharmaceutical agent per hour, day, week ormonth.

“Dosing regimen” is a combination of doses designed to achieve one ormore desired effects.

“Double-stranded antisense compound” means an antisense compoundcomprising two oligomeric compounds that are complementary to each otherand form a duplex, and wherein one of the two said oligomeric compoundscomprises an oligonucleotide.

“Effective amount” means the amount of compound sufficient to effectuatea desired physiological outcome in an individual in need of thecompound. The effective amount may vary among individuals depending onthe health and physical condition of the individual to be treated, thetaxonomic group of the individuals to be treated, the formulation of thecomposition, assessment of the individual's medical condition, and otherrelevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to, the products oftranscription and translation.

“Gapmer” means an oligonucleotide comprising an internal region having aplurality of nucleosides that support RNase H cleavage positionedbetween external regions having one or more nucleosides, wherein thenucleosides comprising the internal region are chemically distinct fromthe nucleoside or nucleosides comprising the external regions. Theinternal region may be referred to as the “gap” and the external regionsmay be referred to as the “wings.”

“Hybridization” means the annealing of oligonucleotides and/or nucleicacids. While not limited to a particular mechanism, the most commonmechanism of hybridization involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases. In certain embodiments, complementary nucleicacid molecules include, but are not limited to, an antisense compoundand a nucleic acid target. In certain embodiments, complementary nucleicacid molecules include, but are not limited to, an oligonucleotide and anucleic acid target.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements of the same kind (e.g. no interveningnucleobases between the immediately adjacent nucleobases).

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity relative to the expression ofactivity in an untreated or control sample and does not necessarilyindicate a total elimination of expression or activity.

“Internucleoside linkage” means a group or bond that forms a covalentlinkage between adjacent nucleosides in an oligonucleotide. “Modifiedinternucleoside linkage” means any internucleoside linkage other than anaturally occurring, phosphate internucleoside linkage. Non-phosphatelinkages are referred to herein as modified internucleoside linkages.

“Lengthened oligonucleotides” are those that have one or more additionalnucleosides relative to an oligonucleotide disclosed herein, e.g. aparent oligonucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Linker-nucleoside” means a nucleoside that links an oligonucleotide toa conjugate moiety. Linker-nucleosides are located within the conjugatelinker of a compound. Linker-nucleosides are not considered part of theoligonucleotide portion of a compound even if they are contiguous withthe oligonucleotide.

“Mismatch” or “non-complementary” means a nucleobase of a firstoligonucleotide that is not complementary to the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligonucleotides are aligned. For example, nucleobasesincluding but not limited to a universal nucleobase, inosine, andhypoxanthine, are capable of hybridizing with at least one nucleobasebut are still mismatched or non-complementary with respect to nucleobaseto which it hybridized. As another example, a nucleobase of a firstoligonucleotide that is not capable of hybridizing to the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligonucleotides are aligned is a mismatch ornon-complementary nucleobase.

“Modulating” refers to changing or adjusting a feature in a cell,tissue, organ or organism. For example, modulating PNPLA3 RNA can meanto increase or decrease the level of PNPLA3 RNA and/or PNPLA3 protein ina cell, tissue, organ or organism. A “modulator” effects the change inthe cell, tissue, organ or organism. For example, a PNPLA3 compound canbe a modulator that decreases the amount of PNPLA3 RNA and/or PNPLA3protein in a cell, tissue, organ or organism.

“MOE” means methoxyethyl.

“Monomer” refers to a single unit of an oligomer. Monomers include, butare not limited to, nucleosides and nucleotides.

“Motif” means the pattern of unmodified and/or modified sugar moieties,nucleobases, and/or internucleoside linkages, in an oligonucleotide.

“Natural” or “naturally occurring” means found in nature.

“Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety”means a modified sugar moiety that comprises a modification, such as asubstituent, that does not form a bridge between two atoms of the sugarto form a second ring.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids, anddouble-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid. As used herein a “naturally occurringnucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), andguanine (G). A “modified nucleobase” is a naturally occurring nucleobasethat is chemically modified. A “universal base” or “universalnucleobase” is a nucleobase other than a naturally occurring nucleobaseand modified nucleobase, and is capable of pairing with any nucleobase.

“Nucleobase sequence” means the order of contiguous nucleobases in anucleic acid or oligonucleotide independent of any sugar orinternucleoside linkage.

“Nucleoside” means a compound comprising a nucleobase and a sugarmoiety. The nucleobase and sugar moiety are each, independently,unmodified or modified. “Modified nucleoside” means a nucleosidecomprising a modified nucleobase and/or a modified sugar moiety.Modified nucleosides include abasic nucleosides, which lack anucleobase.

“Oligomeric compound” means a compound comprising a singleoligonucleotide and optionally one or more additional features, such asa conjugate group or terminal group.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another. Unlessotherwise indicated, oligonucleotides consist of 8-80 linkednucleosides. “Modified oligonucleotide” means an oligonucleotide,wherein at least one sugar, nucleobase, or internucleoside linkage ismodified. “Unmodified oligonucleotide” means an oligonucleotide thatdoes not comprise any sugar, nucleobase, or internucleosidemodification.

“Parent oligonucleotide” means an oligonucleotide whose sequence is usedas the basis of design for more oligonucleotides of similar sequence butwith different lengths, motifs, and/or chemistries. The newly designedoligonucleotides may have the same or overlapping sequence as the parentoligonucleotide.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration.

“Pharmaceutically acceptable carrier or diluent” means any substancesuitable for use in administering to an individual. For example, apharmaceutically acceptable carrier can be a sterile aqueous solution,such as PBS or water-for-injection.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of compounds, such as oligomericcompounds or oligonucleotides, i.e., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto.

“Pharmaceutical agent” means a compound that provides a therapeuticbenefit when administered to an individual.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more compounds or salt thereof and asterile aqueous solution.

“Phosphorothioate linkage” means a modified phosphate linkage in whichone of the non-bridging oxygen atoms is replaced with a sulfur atom. Aphosphorothioate internucleoside linkage is a modified internucleosidelinkage.

“Phosphorus moiety” means a group of atoms comprising a phosphorus atom.In certain embodiments, a phosphorus moiety comprises a mono-, di-, ortri-phosphate, or phosphorothioate.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an oligomeric compound.

“Prevent” refers to delaying or forestalling the onset, development orprogression of a disease, disorder, or condition for a period of timefrom minutes to indefinitely.

“Prodrug” means a compound in a form outside the body which, whenadministered to an individual, is metabolized to another form within thebody or cells thereof. In certain embodiments, the metabolized form isthe active, or more active, form of the compound (e.g., drug). Typicallyconversion of a prodrug within the body is facilitated by the action ofan enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) presentin cells or tissues, and/or by physiologic conditions.

“Reduce” means to bring down to a smaller extent, size, amount, ornumber.

“RefSeq No.” is a unique combination of letters and numbers assigned toa sequence to indicate the sequence is for a particular targettranscript (e.g., target gene). Such sequence and information about thetarget gene (collectively, the gene record) can be found in a geneticsequence database. Genetic sequence databases include the NCBI ReferenceSequence database, GenBank, the European Nucleotide Archive, and the DNAData Bank of Japan (the latter three forming the InternationalNucleotide Sequence Database Collaboration or INSDC).

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“RNAi compound” means an antisense compound that acts, at least in part,through RISC or Ago2, but not through RNase H, to modulate a targetnucleic acid and/or protein encoded by a target nucleic acid. RNAicompounds include, but are not limited to double-stranded siRNA,single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.

“Segments” are defined as smaller or sub-portions of regions within anucleic acid.

“Side effects” means physiological disease and/or conditionsattributable to a treatment other than the desired effects. In certainembodiments, side effects include injection site reactions, liverfunction test abnormalities, renal function abnormalities, livertoxicity, renal toxicity, central nervous system abnormalities,myopathies, and malaise. For example, increased aminotransferase levelsin serum may indicate liver toxicity or liver function abnormality. Forexample, increased bilirubin may indicate liver toxicity or liverfunction abnormality.

“Single-stranded” in reference to a compound means the compound has onlyone oligonucleotide. “Self-complementary” means an oligonucleotide thatat least partially hybridizes to itself. A compound consisting of oneoligonucleotide, wherein the oligonucleotide of the compound isself-complementary, is a single-stranded compound. A single-strandedcompound may be capable of binding to a complementary compound to form aduplex.

“Sites” are defined as unique nucleobase positions within a targetnucleic acid.

“Specifically hybridizable” refers to an oligonucleotide having asufficient degree of complementarity between the oligonucleotide and atarget nucleic acid to induce a desired effect, while exhibiting minimalor no effects on non-target nucleic acids. In certain embodiments,specific hybridization occurs under physiological conditions.

“Specifically inhibit” with reference to a target nucleic acid means toreduce or block expression of the target nucleic acid while exhibitingfewer, minimal, or no effects on non-target nucleic acids. Reductiondoes not necessarily indicate a total elimination of the target nucleicacid's expression.

“Standard cell assay” means assay(s) described in the Examples andreasonable variations thereof

“Standard in vivo experiment” means the procedure(s) described in theExample(s) and reasonable variations thereof.

“Stereorandom chiral center” in the context of a population of moleculesof identical molecular formula means a chiral center having a randomstereochemical configuration. For example, in a population of moleculescomprising a stereorandom chiral center, the number of molecules havingthe (S) configuration of the stereorandom chiral center may be but isnot necessarily the same as the number of molecules having the (R)configuration of the stereorandom chiral center. The stereochemicalconfiguration of a chiral center is considered random when it is theresult of a synthetic method that is not designed to control thestereochemical configuration. In certain embodiments, a stereorandomchiral center is a stereorandom phosphorothioate internucleosidelinkage.

“Sugar moiety” means an unmodified sugar moiety or a modified sugarmoiety. “Unmodified sugar moiety” or “unmodified sugar” means a 2′-OH(H)ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a2′-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).“Modified sugar moiety” or “modified sugar” means a modified furanosylsugar moiety or a sugar surrogate. “Modified furanosyl sugar moiety”means a furanosyl sugar comprising a non-hydrogen substituent in placeof at least one hydrogen or hydroxyl of an unmodified sugar moiety. Incertain embodiments, a modified furanosyl sugar moiety is a2′-substituted sugar moiety. Such modified furanosyl sugar moietiesinclude bicyclic sugars and non-bicyclic sugars.

“Sugar surrogate” means a modified sugar moiety having other than afuranosyl moiety that can link a nucleobase to another group, such as aninternucleoside linkage, conjugate group, or terminal group in anoligonucleotide. Modified nucleosides comprising sugar surrogates can beincorporated into one or more positions within an oligonucleotide andsuch oligonucleotides are capable of hybridizing to complementarycompounds or nucleic acids.

“Synergy” or “synergize” refers to an effect of a combination that isgreater than additive of the effects of each component alone at the samedoses.

“PNPLA3” means any nucleic acid or protein of PNPLA3. “PNPLA3 nucleicacid” means any nucleic acid encoding PNPLA3. For example, in certainembodiments, a PNPLA3 nucleic acid includes a DNA sequence encodingPNPLA3, an RNA sequence transcribed from DNA encoding PNPLA3 (includinggenomic DNA comprising introns and exons), and an mRNA sequence encodingPNPLA3. “PNPLA3 mRNA” means an mRNA encoding a PNPLA3 protein. Thetarget may be referred to in either upper or lower case.

“PNPLA3 specific inhibitor” refers to any agent capable of specificallyinhibiting PNPLA3 RNA and/or PNPLA3 protein expression or activity atthe molecular level. For example, PNPLA3 specific inhibitors includenucleic acids (including antisense compounds), peptides, antibodies,small molecules, and other agents capable of inhibiting the expressionof PNPLA3 RNA and/or PNPLA3 protein.

“Target gene” refers to a gene encoding a target.

“Targeting” means the specific hybridization of a compound to a targetnucleic acid in order to induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby compounds described herein.

“Target region” means a portion of a target nucleic acid to which one ormore compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which a compound is targeted. “5′ target site” refers to the5′-most nucleotide of a target segment. “3′ target site” refers to the3′-most nucleotide of a target segment.

“Terminal group” means a chemical group or group of atoms that iscovalently linked to a terminus of an oligonucleotide.

“Therapeutically effective amount” means an amount of a compound,pharmaceutical agent, or composition that provides a therapeutic benefitto an individual.

“Treat” refers to administering a compound or pharmaceutical compositionto an animal in order to effect an alteration or improvement of adisease, disorder, or condition in the animal.

Certain Embodiments

Certain embodiments provide methods, compounds and compositions forinhibiting PNPLA3 (PNPLA3) expression.

Certain embodiments provide compounds targeted to a PNPLA3 nucleic acid.In certain embodiments, the PNPLA3 nucleic acid has the sequence setforth in RefSeq or GENBANK Accession No. NM_025225.2 (incorporated byreference, disclosed herein as SEQ ID NO: 1); NC_000022.11 truncatedfrom nucleotides 43921001 to 43954500 (incorporated by reference,disclosed herein as SEQ ID NO: 2); AK123806.1(incorporated by reference,disclosed herein as SEQ ID NO: 3); BQ686328.1 (incorporated byreference, disclosed herein as SEQ ID NO: 4); BF762711.1 (incorporatedby reference, disclosed herein as SEQ ID NO: 5); DA290491.1(incorporated by reference, disclosed herein as SEQ ID NO: 6); and thesequences listed as SEQ ID Nos 7, 8, 9, and 10. In certain embodiments,the compound is an antisense compound or oligomeric compound. In certainembodiments, the compound is single-stranded. In certain embodiments,the compound is double-stranded.

In certain embodiments, the compound comprises a modifiedoligonucleotide 16 linked nucleosides in length. In certain embodiments,the compound is an antisense compound or oligomeric compound.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 12 to 30 linked nucleosides in length and having anucleobase sequence comprising the nucleobase sequence of any one of SEQID NOs: 17-2169. In certain embodiments, the compound is an antisensecompound or oligomeric compound. In certain embodiments, the compound issingle-stranded. In certain embodiments, the compound isdouble-stranded. In certain embodiments, the modified oligonucleotide is16 to 30 linked nucleosides in length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide consisting of the nucleobase sequence of any one of SEQID NOs: 17-2169. In certain embodiments, the compound is an antisensecompound or oligomeric compound. In certain embodiments, the compound issingle-stranded. In certain embodiments, the compound isdouble-stranded.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 12 to 30 linked nucleosides in length and complementarywithin nucleobases 5567-5642, 5644-5731, 5567-5731, 5567-5620,13697-13733, 20553-20676, 20664-20824, 20553-20824, and 25844-25912 ofSEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, atleast 90%, at least 95%, or 100% complementary to SEQ ID NO: 2. Incertain embodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 16 to 30 linked nucleosidesin length.

In certain embodiments, compounds target nucleotides 5567-5620 of aPNPLA3 nucleic acid. In certain embodiments, compounds target withinnucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of a PNPLA3nucleic acid having the nucleobase sequence of SEQ ID NO: 2. In certainembodiments, compounds have at least an 8, 9, 10, 11, 12, 13, 14, 15, or16 contiguous nucleobase portion complementary to an equal lengthportion within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 ofa PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO: 2. Incertain embodiments, these compounds are antisense compounds, oligomericcompounds, or oligonucleotides.

In certain embodiments, a compound comprises a modified oligonucleotide12 to 30 linked nucleosides in length and having a nucleobase sequencecomprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguousnucleobase portion any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330,1665, 408, 830, and 899. In certain embodiments, the modifiedoligonucleotide is 16 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide12 to 30 linked nucleosides in length and having a nucleobase sequencecomprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408,830, and 899. In certain embodiments, the modified oligonucleotide is 16to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotidehaving a nucleobase sequence consisting of any one of SEQ ID NOs: 1089,1757, 141, 1982, 330, 1665, 408, 830, and 899.

In certain embodiments, compounds targeted to PNPLA3 is ION 975616,994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Outof over 2,384 compounds that were screened as described in the Examplessection below, ION 975616, 994284, 975605, 994282, 975613, 975617,975735, 975736, and 975612 emerged as the top lead compounds.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified internucleoside linkage, at least onemodified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified sugar. In certain embodiments, at leastone modified sugar comprises a 2′-O-methoxyethyl group. In certainembodiments, at least one modified sugar is a bicyclic sugar, such as a4′-CH(CH3)-O-2′ group, a 4′-CH2-O-2′ group, or a 4′-(CH2)2-O-2′ group.

In certain embodiments, the modified oligonucleotide comprises at leastone modified internucleoside linkage, such as a phosphorothioateinternucleoside linkage.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises:

-   -   a gap segment consisting of linked deoxynucleosides;    -   a 5′ wing segment consisting of linked nucleosides; and    -   a 3′ wing segment consisting of linked nucleosides;        wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar. In certain        embodiments, the modified oligonucleotide is 12 to 30 linked        nucleosides in length having a nucleobase sequence comprising        the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141,        1982, 330, 1665, 408, 830, and 899. In certain embodiments, the        modified oligonucleotide is 16 to 30 linked nucleosides in        length having a nucleobase sequence comprising the sequence        recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330,        1665, 408, 830, and 899. In certain embodiments, the modified        oligonucleotide is 16 linked nucleosides in length having a        nucleobase sequence consisting of the sequence recited in any        one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830,        and 899.

In certain embodiments, a compound comprises or consists of a modifiedoligonucleotide 12-30 linked nucleobases in length having a nucleobasesequence comprising the sequence recited in any one of SEQ ID NOs: 1089,1757, 141, 1982, 330, 1665, 408, 830, and 899, wherein the modifiedoligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a cEt sugar; wherein each internucleoside linkage is aphosphorothioate linkage and wherein each cytosine is a5-methylcytosine. In certain embodiments, the modified oligonucleotideconsists of 16-30 linked nucleosides. In certain embodiments, themodified oligonucleotide consists of 16 linked nucleosides.

In certain embodiments, a compound comprises or consists of a modifiedoligonucleotide, wherein the modified oligonucleotide is 16 linkednucleosides in length and consists of the sequence of SEQ ID NO: 1089,wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a cEt sugar; wherein each internucleoside linkage is aphosphorothioate linkage; and wherein each cytosine is a5-methylcytosine.

In certain embodiments, a compound consists of a modifiedoligonucleotide and a conjugate group, wherein the modifiedoligonucleotide is 16 linked nucleosides in length and consists of thesequence of SEQ ID NO: 1089, wherein the modified oligonucleotidecomprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a cEt sugar; wherein each internucleoside linkage is aphosphorothioate linkage; wherein each cytosine is a 5-methylcytosine;and wherein the conjugate group is positioned at the 5′end of themodified oligonucleotide and is

In certain embodiments, a compound comprises or consists of ION 916333or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975616or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975616, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975613or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975613, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975612or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975612, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 916789or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 916789, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 916602or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 916602, having the following chemical structure:

In any of the foregoing embodiments, the compound or oligonucleotide canbe at least 85%, at least 90%, at least 95%, at least 98%, at least 99%,or 100% complementary to a nucleic acid encoding PNPLA3.

In any of the foregoing embodiments, the compound can besingle-stranded. In certain embodiments, the compound comprisesdeoxyribonucleotides. In certain embodiments, the compound isdouble-stranded. In certain embodiments, the compound is double-strandedand comprises ribonucleotides. In any of the foregoing embodiments, thecompound can be an antisense compound or oligomeric compound.

In any of the foregoing embodiments, the compound can be 8 to 80, 10 to30, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linkednucleosides in length. In certain embodiments, the compound comprises orconsists of an oligonucleotide.

In certain embodiments, a compound comprises a modified oligonucleotidedescribed herein and a conjugate group. In certain embodiments, theconjugate group is linked to the modified oligonucleotide at the 5′ endof the modified oligonucleotide. In certain embodiments, the conjugategroup is linked to the modified oligonucleotide at the 3′ end of themodified oligonucleotide. In certain embodiments, the conjugate groupcomprises at least one N-Acetylgalactosamine (GalNAc), at least twoN-Acetylgalactosamines (GalNAcs), or at least threeN-Acetylgalactosamines (GalNAcs).

In certain embodiments, compounds or compositions provided hereincomprise a pharmaceutically acceptable salt of the modifiedoligonucleotide. In certain embodiments, the salt is a sodium salt. Incertain embodiments, the salt is a potassium salt.

In certain embodiments, the compounds or compositions as describedherein are active by virtue of having at least one of an in vitro IC₅₀of less than 2 μM, less than 1.5 μM, less than 1 μM, less than 0.9 μM,less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM,less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM,less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02μM, or less than 0.01 μM.

In certain embodiments, the compounds or compositions as describedherein are highly tolerable as demonstrated by having at least one of anincrease in alanine transaminase (ALT) or aspartate transaminase (AST)value of no more than 4 fold, 3 fold, or 2 fold over control animals, oran increase in liver, spleen, or kidney weight of no more than 30%, 20%,15%, 12%, 10%, 5%, or 2% compared to control animals. In certainembodiments, the compounds or compositions as described herein arehighly tolerable as demonstrated by having no increase of ALT or ASTover control animals. In certain embodiments, the compounds orcompositions as described herein are highly tolerable as demonstrated byhaving no increase in liver, spleen, or kidney weight over controlanimals.

Certain embodiments provide a composition comprising the compound of anyof the aforementioned embodiments or any pharmaceutically acceptablesalt thereof and at least one of a pharmaceutically acceptable carrieror diluent. In certain embodiments, the composition has a viscosity lessthan about 40 centipoise (cP), less than about 30 centipose (cP), lessthan about 20 centipose (cP), less than about 15 centipose (cP), or lessthan about 10 centipose (cP). In certain embodiments, the compositionhaving any of the aforementioned viscosities comprises a compoundprovided herein at a concentration of about 100 mg/mL, about 125 mg/mL,about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL,about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certainembodiments, the composition having any of the aforementionedviscosities and/or compound concentrations has a temperature of roomtemperature, or about 20° C., about 21° C., about 22° C., about 23° C.,about 24° C., about 25° C., about 26° C., about 27° C., about 28° C.,about 29° C., or about 30° C.

Certain Indications

Certain embodiments provided herein relate to methods of inhibitingPNPLA3 expression, which can be useful for treating, preventing, orameliorating a disease associated with PNPLA3 in an individual, byadministration of a compound that targets PNPLA3. In certainembodiments, the compound can be a PNPLA3 specific inhibitor. In certainembodiments, the compound can be an antisense compound, an oligomericcompound, or an oligonucleotide targeted to PNPLA3.

Examples of diseases associated with PNPLA3 treatable, preventable,and/or ameliorable with the methods provided herein include liverdisease, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH),liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease,alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis,hereditary hemochromatosis, or primary sclerosing cholangitis. Certaincompounds provided herein are directed to compounds and compositionsthat reduce liver damage, steatosis, liver fibrosis, liver inflammation,liver scarring or cirrhosis, liver failure, liver enlargement, elevatedtransaminases, or hepatic fat accumulation in an animal.

In certain embodiments, a method of treating, preventing, orameliorating a disease associated with PNPLA3 in an individual comprisesadministering to the individual a compound comprising a PNPLA3 specificinhibitor, thereby treating, preventing, or ameliorating the disease. Incertain embodiments, the individual is identified as having, or at riskof having, a disease associated with PNPLA3. In certain embodiments, thedisease is a liver disease. In certain embodiments, the compoundcomprises an antisense compound targeted to PNPLA3. In certainembodiments, the compound comprises an oligonucleotide targeted toPNPLA3. In certain embodiments, a compound comprises a modifiedoligonucleotide 12 to 30 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 12 to 30linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 16 to 30linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.In certain embodiments, a compound comprises a modified oligonucleotidehaving a nucleobase sequence consisting of any one of SEQ ID NOs: 1089,1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments,the compound is ION 975616, 994284, 975605, 994282, 975613, 975617,975735, 975736, or 975612. In any of the foregoing embodiments, thecompound can be single-stranded or double-stranded. In any of theforegoing embodiments, the compound can be an antisense compound oroligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments,administering the compound improves, preserves, or prevents liverdamage, steatosis, liver fibrosis, cirrhosis, elevated transaminases, orhepatic fat accumulation in an animal.

In certain embodiments, a method of treating, preventing, orameliorating liver damage, steatosis, liver fibrosis, liverinflammation, liver scarring or cirrhosis, liver failure, liverenlargement, elevated transaminases, or hepatic fat accumulation in ananimal comprises administering to the individual a compound comprising aPNPLA3 specific inhibitor, thereby treating, preventing, or amelioratingliver damage, steatosis, liver fibrosis, liver inflammation, liverscarring or cirrhosis, liver failure, liver enlargement, elevatedtransaminases, or hepatic fat accumulation. In certain embodiments, thecompound comprises an antisense compound targeted to PNPLA3. In certainembodiments, the compound comprises an oligonucleotide targeted toPNPLA3. In certain embodiments, a compound comprises a modifiedoligonucleotide 12 to 30 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 12 to 30linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 16 to 30linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.In certain embodiments, a compound comprises a modified oligonucleotidehaving a nucleobase sequence consisting of any one of SEQ ID NOs: 1089,1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments,the compound is ION 975616, 994284, 975605, 994282, 975613, 975617,975735, 975736, or 975612. In any of the foregoing embodiments, thecompound can be single-stranded or double-stranded. In any of theforegoing embodiments, the compound can be an antisense compound oroligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments,administering the compound improves, preserves, or prevents liverdamage, steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation. In certain embodiments, the individual isidentified as having, or at risk of having, a disease associated withPNPLA3.

In certain embodiments, a method of inhibiting expression of PNPLA3 inan individual having, or at risk of having, a disease associated withPNPLA3 comprises administering to the individual a compound comprising aPNPLA3 specific inhibitor, thereby inhibiting expression of PNPLA3 inthe individual. In certain embodiments, administering the compoundinhibits expression of PNPLA3 in the liver. In certain embodiments, thedisease is a liver disease. In certain embodiments, the individual has,or is at risk of having, NAFLD, hepatic steatosis, non-alcoholicsteatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma,alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis,chronic hepatitis, hereditary hemochromatosis, or primary sclerosingcholangitis. In certain embodiments, the individual has, or is at riskof having, liver damage, steatosis, liver fibrosis, liver inflammation,liver scarring or cirrhosis, liver failure, liver enlargement, elevatedtransaminases, or hepatic fat accumulation. In certain embodiments, thecompound comprises an antisense compound targeted to PNPLA3. In certainembodiments, the compound comprises an oligonucleotide targeted toPNPLA3. In certain embodiments, a compound comprises a modifiedoligonucleotide 12 to 30 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 12 to 30linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certainembodiments, a compound comprises a modified oligonucleotide 16 to 30linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.In certain embodiments, a compound comprises a modified oligonucleotidehaving a nucleobase sequence consisting of any one of SEQ ID NOs: 1089,1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments,the compound is ION 975616, 994284, 975605, 994282, 975613, 975617,975735, 975736, or 975612. In any of the foregoing embodiments, thecompound can be single-stranded or double-stranded. In any of theforegoing embodiments, the compound can be an antisense compound oroligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments,administering the compound improves, preserves, or prevents liverdamage, steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation.

In certain embodiments, a method of inhibiting expression of PNPLA3 in acell comprises contacting the cell with a compound comprising a PNPLA3specific inhibitor, thereby inhibiting expression of PNPLA3 in the cell.In certain embodiments, the cell is a hepatocyte. In certainembodiments, the cell is in the liver. In certain embodiments, the cellis in the liver of an individual who has, or is at risk of having, liverdamage, steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation. In certain embodiments, the compound comprisesan antisense compound targeted to PNPLA3. In certain embodiments, thecompound comprises an oligonucleotide targeted to PNPLA3. In certainembodiments, a compound comprises a modified oligonucleotide 12 to 30linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 17-2169. In certain embodiments, a compound comprises amodified oligonucleotide 12 to 30 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprisesa modified oligonucleotide 16 to 30 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141,1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compoundcomprises a modified oligonucleotide having a nucleobase sequenceconsisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665,408, 830, and 899. In certain embodiments, the compound is ION 975616,994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Inany of the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or oligomeric compound.

In certain embodiments, a method of reducing or inhibiting liver damage,steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation in an individual having, or at risk of having,a disease associated with PNPLA3 comprises administering to theindividual a compound comprising a PNPLA3 specific inhibitor, therebyreducing or inhibiting liver damage, steatosis, liver fibrosis, liverinflammation, liver scarring or cirrhosis, liver failure, liverenlargement, elevated transaminases, or hepatic fat accumulation in theindividual. In certain embodiments, the individual has, or is at risk ofhaving, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH),liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease,alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis,hereditary hemochromatosis, or primary sclerosing cholangitis. Incertain embodiments, the compound comprises an antisense compoundtargeted to PNPLA3. In certain embodiments, the compound comprises anoligonucleotide targeted to PNPLA3. In certain embodiments, a compoundcomprises a modified oligonucleotide 12 to 30 linked nucleosides inlength and having a nucleobase sequence comprising at least 8 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide12 to 30 linked nucleosides in length and having a nucleobase sequencecomprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. Incertain embodiments, a compound comprises a modified oligonucleotideconsisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide16 to 30 linked nucleosides in length having a nucleobase sequencecomprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408,830, and 899. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. Incertain embodiments, the compound is ION 975616, 994284, 975605, 994282,975613, 975617, 975735, 975736, or 975612. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments, theindividual is identified as having, or at risk of having, a diseaseassociated with PNPLA3.

Certain embodiments are drawn to a compound comprising a PNPLA3 specificinhibitor for use in treating a disease associated with PNPLA3. Incertain embodiments, the disease is NAFLD, hepatic steatosis,non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellularcarcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCVhepatitis, chronic hepatitis, hereditary hemochromatosis, or primarysclerosing cholangitis. In certain embodiments, the compound comprisesan antisense compound targeted to PNPLA3. In certain embodiments, thecompound comprises an oligonucleotide targeted to PNPLA3. In certainembodiments, a compound comprises a modified oligonucleotide 12 to 30linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 17-2169. In certain embodiments, a compound comprises amodified oligonucleotide 12 to 30 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprisesa modified oligonucleotide 16 to 30 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141,1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compoundcomprises a modified oligonucleotide having a nucleobase sequenceconsisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665,408, 830, and 899. In certain embodiments, the compound is ION 975616,994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Inany of the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or oligomeric compound. In certain embodiments,the compound is administered to the individual parenterally.

Certain embodiments are drawn to a compound comprising a PNPLA3 specificinhibitor for use in reducing or inhibiting liver damage, steatosis,liver fibrosis, liver inflammation, liver scarring or cirrhosis, liverfailure, liver enlargement, elevated transaminases, or hepatic fataccumulation in an individual having, or at risk of having, NAFLD,hepatic steatosis, non-alcoholic steatohepatitis (NASH), livercirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholicsteatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditaryhemochromatosis, or primary sclerosing cholangitis. In certainembodiments, the compound comprises an antisense compound targeted toPNPLA3. In certain embodiments, the compound comprises anoligonucleotide targeted to PNPLA3. In certain embodiments, a compoundcomprises a modified oligonucleotide 12 to 30 linked nucleosides inlength and having a nucleobase sequence comprising at least 8 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide12 to 30 linked nucleosides in length and having a nucleobase sequencecomprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. Incertain embodiments, a compound comprises a modified oligonucleotideconsisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide16 to 30 linked nucleosides in length having a nucleobase sequencecomprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408,830, and 899. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. Incertain embodiments, the compound is ION 975616, 994284, 975605, 994282,975613, 975617, 975735, 975736, or 975612. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound.

Certain embodiments are drawn to the use of a compound comprising aPNPLA3 specific inhibitor for the manufacture or preparation of amedicament for treating a disease associated with PNPLA3. Certainembodiments are drawn to the use of a compound comprising a PNPLA3specific inhibitor for the preparation of a medicament for treating adisease associated with PNPLA3. In certain embodiments, the disease is aliver disease. In certain embodiments, the disease is NAFLD, hepaticsteatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis,hepatocellular carcinoma, alcoholic liver disease, alcoholicsteatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditaryhemochromatosis, or primary sclerosing cholangitis. In certainembodiments, the compound comprises an antisense compound targeted toPNPLA3. In certain embodiments, the compound comprises anoligonucleotide targeted to PNPLA3. In certain embodiments, a compoundcomprises a modified oligonucleotide 12 to 30 linked nucleosides inlength and having a nucleobase sequence comprising at least 8 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide12 to 30 linked nucleosides in length and having a nucleobase sequencecomprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. Incertain embodiments, a compound comprises a modified oligonucleotideconsisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169.In certain embodiments, a compound comprises a modified oligonucleotide16 to 30 linked nucleosides in length having a nucleobase sequencecomprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408,830, and 899. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. Incertain embodiments, the compound is ION 975616, 994284, 975605, 994282,975613, 975617, 975735, 975736, or 975612. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or an oligomeric compound.

Certain embodiments are drawn to the use of a compound comprising aPNPLA3 specific inhibitor for the manufacture or preparation of amedicament for reducing or inhibiting liver damage, steatosis, liverfibrosis, liver inflammation, liver scarring or cirrhosis, liverfailure, liver enlargement, elevated transaminases, or hepatic fataccumulation in an individual having, or at risk of having, a liverdisease associated with PNPLA3. In certain embodiments, the liverdisease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis(NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liverdisease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronichepatitis, hereditary hemochromatosis, or primary sclerosingcholangitis. Certain embodiments are drawn to use of a compoundcomprising a PNPLA3 specific inhibitor for the preparation of amedicament for treating a disease associated with PNPLA3. In certainembodiments, the disease is NAFLD, hepatic steatosis, non-alcoholicsteatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma,alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis,chronic hepatitis, hereditary hemochromatosis, or primary sclerosingcholangitis. In certain embodiments, the compound comprises an antisensecompound targeted to PNPLA3. In certain embodiments, the compoundcomprises an oligonucleotide targeted to PNPLA3. In certain embodiments,a compound comprises a modified oligonucleotide 12 to 30 linkednucleosides in length and having a nucleobase sequence comprising atleast 8 contiguous nucleobases of any of the nucleobase sequences of SEQID NOs: 17-2169. In certain embodiments, a compound comprises a modifiedoligonucleotide 12 to 30 linked nucleosides in length and having anucleobase sequence comprising the nucleobase sequence of any one of SEQID NOs: 17-2169. In certain embodiments, a compound comprises a modifiedoligonucleotide consisting of the nucleobase sequence of any one of SEQID NOs: 17-2169. In certain embodiments, a compound comprises a modifiedoligonucleotide 16 to 30 linked nucleosides in length having anucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141,1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compoundcomprises a modified oligonucleotide having a nucleobase sequenceconsisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665,408, 830, and 899. In certain embodiments, the compound is ION 975616,994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Inany of the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or an oligomeric compound.

In any of the foregoing methods or uses, the compound can be targeted toPNPLA3. In certain embodiments, the compound comprises or consists of amodified oligonucleotide, for example, a modified oligonucleotide 8 to80 linked nucleosides in length, 10 to 30 linked nucleosides in length,12 to 30 linked nucleosides in length, or 20 linked nucleosides inlength. In certain embodiments, the modified oligonucleotide is at least80%, at least 85%, at least 90%, at least 95% or 100% complementary toany of the nucleobase sequences recited in SEQ ID NOs: 1-10. In certainembodiments, the modified oligonucleotide comprises at least onemodified internucleoside linkage, at least one modified sugar and/or atleast one modified nucleobase. In certain embodiments, the modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage,the modified sugar is a bicyclic sugar or a 2′-O-methoxyethyl modifiedsugar, and the modified nucleobase is a 5-methylcytosine. In certainembodiments, the modified oligonucleotide comprises a gap segmentconsisting of linked deoxynucleosides; a 5′ wing segment consisting oflinked nucleosides; and a 3′ wing segment consisting of linkednucleosides, wherein the gap segment is positioned immediately adjacentto and between the 5′ wing segment and the 3′ wing segment and whereineach nucleoside of each wing segment comprises a modified sugar.

In any of the foregoing embodiments, the modified oligonucleotide is 12to 30, 15 to 30, 15 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to24, 20 to 24, 19 to 22, 20 to 22, 16 to 20, or 16 or 20 linkednucleosides in length. In certain embodiments, the modifiedoligonucleotide is at least 80%, at least 85%, at least 90%, at least95% or 100% complementary to any of the nucleobase sequences recited inSEQ ID NOs: 1-10.

In any of the foregoing methods or uses, the compound comprises orconsists of a modified oligonucleotide 16 to 30 linked nucleosides inlength and having a nucleobase sequence comprising any one of SEQ IDNOs: 17-2169, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked 2′-deoxynucleosides;    -   a 5′ wing segment consisting of linked nucleosides; and    -   a 3′ wing segment consisting of linked nucleosides;        wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

In any of the foregoing methods or uses, the compound comprises orconsists a modified oligonucleotide 16 linked nucleosides in lengthhaving a nucleobase sequence comprising the sequence recited in any oneof SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899,wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of three linked nucleosides; and

a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a cEt sugar; wherein each internucleoside linkage is aphosphorothioate linkage and wherein each cytosine is a5-methylcytosine. In certain embodiments, the modified oligonucleotideis 16-30 linked nucleosides in length.

In certain embodiments, a compound comprises or consists of ION 916333or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975616or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975616, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975613or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975613, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 975612or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 975612, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 916789or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 916789, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ION 916602or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of 916602, having the following chemical structure:

In any of the foregoing methods or uses, the compound can beadministered parenterally. For example, in certain embodiments thecompound can be administered through injection or infusion. Parenteraladministration includes subcutaneous administration, intravenousadministration, intramuscular administration, intraarterialadministration, intraperitoneal administration, or intracranialadministration, e.g. intrathecal or intracerebroventricularadministration.

Certain Compounds

In certain embodiments, compounds described herein can be antisensecompounds. In certain embodiments, the antisense compound comprises orconsists of an oligomeric compound. In certain embodiments, theoligomeric compound comprises a modified oligonucleotide. In certainembodiments, the modified oligonucleotide has a nucleobase sequencecomplementary to that of a target nucleic acid.

In certain embodiments, a compound described herein comprises orconsists of a modified oligonucleotide. In certain embodiments, themodified oligonucleotide has a nucleobase sequence complementary to thatof a target nucleic acid.

In certain embodiments, a compound or antisense compound issingle-stranded. Such a single-stranded compound or antisense compoundcomprises or consists of an oligomeric compound. In certain embodiments,such an oligomeric compound comprises or consists of an oligonucleotideand optionally a conjugate group. In certain embodiments, theoligonucleotide is an antisense oligonucleotide. In certain embodiments,the oligonucleotide is modified. In certain embodiments, theoligonucleotide of a single-stranded antisense compound or oligomericcompound comprises a self-complementary nucleobase sequence.

In certain embodiments, compounds are double-stranded. Suchdouble-stranded compounds comprise a first modified oligonucleotidehaving a region complementary to a target nucleic acid and a secondmodified oligonucleotide having a region complementary to the firstmodified oligonucleotide. In certain embodiments, the modifiedoligonucleotide is an RNA oligonucleotide. In such embodiments, thethymine nucleobase in the modified oligonucleotide is replaced by auracil nucleobase. In certain embodiments, compound comprises aconjugate group. In certain embodiments, one of the modifiedoligonucleotides is conjugated. In certain embodiments, both themodified oligonucleotides are conjugated. In certain embodiments, thefirst modified oligonucleotide is conjugated. In certain embodiments,the second modified oligonucleotide is conjugated. In certainembodiments, the first modified oligonucleotide is 16-30 linkednucleosides in length and the second modified oligonucleotide is 16-30linked nucleosides in length. In certain embodiments, one of themodified oligonucleotides has a nucleobase sequence comprising at least8 contiguous nucleobases of any of SEQ ID NOs: 17-2169.

In certain embodiments, antisense compounds are double-stranded. Suchdouble-stranded antisense compounds comprise a first oligomeric compoundhaving a region complementary to a target nucleic acid and a secondoligomeric compound having a region complementary to the firstoligomeric compound. The first oligomeric compound of such doublestranded antisense compounds typically comprises or consists of amodified oligonucleotide and optionally a conjugate group. Theoligonucleotide of the second oligomeric compound of such adouble-stranded antisense compound may be modified or unmodified. Eitheror both oligomeric compounds of a double-stranded antisense compound maycomprise a conjugate group. The oligomeric compounds of double-strandedantisense compounds may include non-complementary overhangingnucleosides.

Examples of single-stranded and double-stranded compounds include, butare not limited to, oligonucleotides, siRNAs, microRNA targetingoligonucleotides, and single-stranded RNAi compounds, such as smallhairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNAmimics.

In certain embodiments, a compound described herein has a nucleobasesequence that, when written in the 5′ to 3′ direction, comprises thereverse complement of the target segment of a target nucleic acid towhich it is targeted.

In certain embodiments, a compound described herein comprises anoligonucleotide 12 to 30 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 12to 22 linked subunits in length. In certain embodiments, compounddescribed herein comprises an oligonucleotide 14 to 30 linked subunitsin length. In certain embodiments, compound described herein comprisesan oligonucleotide 14 to 20 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 15to 30 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 15 to 20 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 16 to 30 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 16to 20 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 17 to 30 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 17 to 20 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 18to 30 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 18 to 20 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 20 to 30 linked subunits in length. In other words,such oligonucleotides are 12 to 30 linked subunits, 14 to 30 linkedsubunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18to 30 subunits, 18 to 20 subunits, or 20 to 30 subunits in length,respectively. In certain embodiments, a compound described hereincomprises an oligonucleotide 14 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 16linked subunits in length. In certain embodiments, a compound describedherein comprises an oligonucleotide 17 linked subunits in length. Incertain embodiments, compound described herein comprises anoligonucleotide 18 linked subunits in length. In certain embodiments, acompound described herein comprises an oligonucleotide 19 linkedsubunits in length. In certain embodiments, a compound described hereincomprises an oligonucleotide 20 linked subunits in length. In otherembodiments, a compound described herein comprises an oligonucleotide 8to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits.In certain such embodiments, the compound described herein comprises anoligonucleotide 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 linked subunits in length, or arange defined by any two of the above values. In some embodiments thelinked subunits are nucleotides, nucleosides, or nucleobases.

In certain embodiments, the compound may further comprise additionalfeatures or elements, such as a conjugate group, that are attached tothe oligonucleotide. In certain embodiments, such compounds areantisense compounds. In certain embodiments, such compounds areoligomeric compounds. In embodiments where a conjugate group comprises anucleoside (i.e. a nucleoside that links the conjugate group to theoligonucleotide), the nucleoside of the conjugate group is not countedin the length of the oligonucleotide.

In certain embodiments, compounds may be shortened or truncated. Forexample, a single subunit may be deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated compound targeted to a PNPLA3 nucleic acid mayhave two subunits deleted from the 5′ end, or alternatively, may havetwo subunits deleted from the 3′ end of the compound. Alternatively, thedeleted nucleosides may be dispersed throughout the compound.

When a single additional subunit is present in a lengthened compound,the additional subunit may be located at the 5′ or 3′ end of thecompound. When two or more additional subunits are present, the addedsubunits may be adjacent to each other, for example, in a compoundhaving two subunits added to the 5′ end (5′ addition), or alternatively,to the 3′ end (3′ addition) of the compound. Alternatively, the addedsubunits may be dispersed throughout the compound.

It is possible to increase or decrease the length of a compound, such asan oligonucleotide, and/or introduce mismatch bases without eliminatingactivity (Woolf et al. Proc. Natl. Acad. Sci. USA 1992, 89:7305-7309;Gautschi et al. J. Natl. Cancer Inst. March 2001, 93:463-471; Maher andDolnick Nuc. Acid. Res. 1998, 16:3341-3358). However, seemingly smallchanges in oligonucleotide sequence, chemistry and motif can make largedifferences in one or more of the many properties required for clinicaldevelopment (Seth et al. J. Med. Chem. 2009, 52, 10; Egli et al. J. Am.Chem. Soc. 2011, 133, 16642).

In certain embodiments, compounds described herein are interfering RNAcompounds (RNAi), which include double-stranded RNA compounds (alsoreferred to as short-interfering RNA or siRNA) and single-stranded RNAicompounds (or ssRNA). Such compounds work at least in part through theRISC pathway to degrade and/or sequester a target nucleic acid (thus,include microRNA/microRNA-mimic compounds). As used herein, the termsiRNA is meant to be equivalent to other terms used to describe nucleicacid molecules that are capable of mediating sequence-specific RNAi, forexample, short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term “RNAi” is meant to be equivalent toother terms used to describe sequence-specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics.

In certain embodiments, a compound described herein can comprise any ofthe oligonucleotide sequences targeted to PNPLA3 described herein. Incertain embodiments, the compound can be double-stranded. In certainembodiments, the compound comprises a first strand comprising at leastan 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion ofany one of SEQ ID NOs: 17-2169 and a second strand. In certainembodiments, the compound comprises a first strand comprising thenucleobase sequence of any one of SEQ ID NOs: 17-2169 and a secondstrand. In certain embodiments, the compound comprises ribonucleotidesin which the first strand has uracil (U) in place of thymine (T) in anyone of SEQ ID NOs: 17-2169. In certain embodiments, the compoundcomprises (i) a first strand comprising a nucleobase sequencecomplementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169is targeted, and (ii) a second strand. In certain embodiments, thecompound comprises one or more modified nucleotides in which the 2′position of the sugar contains a halogen (such as fluorine group; 2′-F)or contains an alkoxy group (such as a methoxy group; 2′-OMe). Incertain embodiments, the compound comprises at least one 2′-F sugarmodification and at least one 2′-OMe sugar modification. In certainembodiments, the at least one 2′-F sugar modification and at least one2′-OMe sugar modification are arranged in an alternating pattern for atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 contiguous nucleobases along a strand of the dsRNA compound. Incertain embodiments, the compound comprises one or more linkages betweenadjacent nucleotides other than a naturally-occurring phosphodiesterlinkage. Examples of such linkages include phosphoramide,phosphorothioate, and phosphorodithioate linkages. The compounds mayalso be chemically modified nucleic acid molecules as taught in U.S.Pat. No. 6,673,661. In other embodiments, the compound contains one ortwo capped strands, as disclosed, for example, by WO 00/63364, filedApr. 19, 2000.

In certain embodiments, the first strand of the compound is an siRNAguide strand and the second strand of the compound is an siRNA passengerstrand. In certain embodiments, the second strand of the compound iscomplementary to the first strand. In certain embodiments, each strandof the compound is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosidesin length. In certain embodiments, the first or second strand of thecompound can comprise a conjugate group.

In certain embodiments, a compound described herein can comprise any ofthe oligonucleotide sequences targeted to PNPLA3 described herein. Incertain embodiments, the compound is single stranded. In certainembodiments, such a compound is a single-stranded RNAi (ssRNAi)compound. In certain embodiments, the compound comprises at least an 8,9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion of anyone of SEQ ID NOs: 17-2169. In certain embodiments, the compoundcomprises the nucleobase sequence of any one of SEQ ID NOs: 17-2169. Incertain embodiments, the compound comprises ribonucleotides in whichuracil (U) is in place of thymine (T) in any one of SEQ ID NOs: 17-2169.In certain embodiments, the compound comprises a nucleobase sequencecomplementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169is targeted. In certain embodiments, the compound comprises one or moremodified nucleotides in which the 2′ position in the sugar contains ahalogen (such as fluorine group; 2′-F) or contains an alkoxy group (suchas a methoxy group; 2′-OMe). In certain embodiments, the compoundcomprises at least one 2′-F sugar modification and at least one 2′-OMesugar modification. In certain embodiments, the at least one 2′-F sugarmodification and at least one 2′-OMe sugar modification are arranged inan alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along a strandof the compound. In certain embodiments, the compound comprises one ormore linkages between adjacent nucleotides other than anaturally-occurring phosphodiester linkage. Examples of such linkagesinclude phosphoramide, phosphorothioate, and phosphorodithioatelinkages. The compounds may also be chemically modified nucleic acidmolecules as taught in U.S. Pat. No. 6,673,661. In other embodiments,the compound contains a capped strand, as disclosed, for example, by WO00/63364, filed Apr. 19, 2000. In certain embodiments, the compoundconsists of 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides. Incertain embodiments, the compound can comprise a conjugate group.

Certain Mechanisms

In certain embodiments, compounds described herein comprise or consistof modified oligonucleotides. In certain embodiments, compoundsdescribed herein are antisense compounds. In certain embodiments,compounds comprise oligomeric compounds. In certain embodiments,compounds described herein are capable of hybridizing to a targetnucleic acid, resulting in at least one antisense activity. In certainembodiments, compounds described herein selectively affect one or moretarget nucleic acid. Such compounds comprise a nucleobase sequence thathybridizes to one or more target nucleic acid, resulting in one or moredesired antisense activity and does not hybridize to one or morenon-target nucleic acid or does not hybridize to one or more non-targetnucleic acid in such a way that results in a significant undesiredantisense activity.

In certain antisense activities, hybridization of a compound describedherein to a target nucleic acid results in recruitment of a protein thatcleaves the target nucleic acid. For example, certain compoundsdescribed herein result in RNase H mediated cleavage of the targetnucleic acid. RNase H is a cellular endonuclease that cleaves the RNAstrand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need notbe unmodified DNA. In certain embodiments, compounds described hereinare sufficiently “DNA-like” to elicit RNase H activity. Further, incertain embodiments, one or more non-DNA-like nucleoside in the gap of agapmer is tolerated.

In certain antisense activities, compounds described herein or a portionof the compound is loaded into an RNA-induced silencing complex (RISC),ultimately resulting in cleavage of the target nucleic acid. Forexample, certain compounds described herein result in cleavage of thetarget nucleic acid by Argonaute. Compounds that are loaded into RISCare RNAi compounds. RNAi compounds may be double-stranded (siRNA) orsingle-stranded (ssRNA).

In certain embodiments, hybridization of compounds described herein to atarget nucleic acid does not result in recruitment of a protein thatcleaves that target nucleic acid. In certain such embodiments,hybridization of the compound to the target nucleic acid results inalteration of splicing of the target nucleic acid. In certainembodiments, hybridization of the compound to a target nucleic acidresults in inhibition of a binding interaction between the targetnucleic acid and a protein or other nucleic acid. In certain suchembodiments, hybridization of the compound to a target nucleic acidresults in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid, a change in theratio of splice variants of a nucleic acid or protein, and/or aphenotypic change in a cell or animal.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, compounds described herein comprise or consistof an oligonucleotide comprising a region that is complementary to atarget nucleic acid. In certain embodiments, the target nucleic acid isan endogenous RNA molecule. In certain embodiments, the target nucleicacid encodes a protein. In certain such embodiments, the target nucleicacid is selected from an mRNA and a pre-mRNA, including intronic, exonicand untranslated regions. In certain embodiments, the target RNA is anmRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. Incertain such embodiments, the target region is entirely within anintron. In certain embodiments, the target region spans an intron/exonjunction. In certain embodiments, the target region is at least 50%within an intron.

Nucleotide sequences that encode PNPLA3 include, without limitation, thefollowing: RefSeq or GENBANK Accession Nos. NM_025225.2 (incorporated byreference, disclosed herein as SEQ ID NO: 1); GENBANK Accession No.NC_000022.11 truncated from nucleotides 43921001 to 43954500(incorporated by reference, disclosed herein as SEQ ID NO: 2);AK123806.1(incorporated by reference, disclosed herein as SEQ ID NO: 3);BQ686328.1 (incorporated by reference, disclosed herein as SEQ ID NO:4); BF762711.1 (incorporated by reference, disclosed herein as SEQ IDNO: 5); DA290491.1 (incorporated by reference, disclosed herein as SEQID NO: 6); and the sequences listed as SEQ ID Nos. 7, 8, 9, and 10.

Hybridization

In some embodiments, hybridization occurs between a compound disclosedherein and a PNPLA3 nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Hybridizationconditions are sequence-dependent and are determined by the nature andcomposition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the compounds provided herein are specifically hybridizablewith a PNPLA3 nucleic acid.

Complementarity

An oligonucleotide is said to be complementary to another nucleic acidwhen the nucleobase sequence of such oligonucleotide or one or moreregions thereof matches the nucleobase sequence of anotheroligonucleotide or nucleic acid or one or more regions thereof when thetwo nucleobase sequences are aligned in opposing directions. Nucleobasematches or complementary nucleobases, as described herein, are limitedto the following pairs: adenine (A) and thymine (T), adenine (A) anduracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (mC) andguanine (G), unless otherwise specified. Complementary oligonucleotidesand/or nucleic acids need not have nucleobase complementarity at eachnucleoside and may include one or more nucleobase mismatches. Anoligonucleotide is fully complementary or 100% complementary when sucholigonucleotides have nucleobase matches at each nucleoside without anynucleobase mismatches.

In certain embodiments, compounds described herein comprise or consistof modified oligonucleotides. In certain embodiments, compoundsdescribed herein are antisense compounds. In certain embodiments,compounds comprise oligomeric compounds. Non-complementary nucleobasesbetween a compound and a PNPLA3 nucleic acid may be tolerated providedthat the compound remains able to specifically hybridize to a targetnucleic acid. Moreover, a compound may hybridize over one or moresegments of a PNPLA3 nucleic acid such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In certain embodiments, the compounds provided herein, or a specifiedportion thereof are at least, or are up to 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a PNPLA3 nucleic acid, a target region, target segment,or specified portion thereof. In certain embodiments, the compoundsprovided herein, or a specified portion thereof, are 70% to 75%, 75% to80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, or any number inbetween these ranges, complementary to a PNPLA3 nucleic acid, a targetregion, target segment, or specified portion thereof Percentcomplementarity of a compound with a target nucleic acid can bedetermined using routine methods.

For example, a compound in which 18 of 20 nucleobases of the compoundare complementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining non-complementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, a compound whichis 18 nucleobases in length having four non-complementary nucleobaseswhich are flanked by two regions of complete complementarity with thetarget nucleic acid would have 77.8% overall complementarity with thetarget nucleic acid. Percent complementarity of a compound with a regionof a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,sequence identity or complementarity, can be determined by, for example,the Gap program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, Madison Wis.),using default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, compounds described herein, or specifiedportions thereof, are fully complementary (i.e. 100% complementary) to atarget nucleic acid, or specified portion thereof. For example, acompound may be fully complementary to a PNPLA3 nucleic acid, or atarget region, or a target segment or target sequence thereof. As usedherein, “fully complementary” means each nucleobase of a compound iscomplementary to the corresponding nucleobase of a target nucleic acid.For example, a 20 nucleobase compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the compound. “Fully complementary” can also beused in reference to a specified portion of the first and/or the secondnucleic acid. For example, a 20 nucleobase portion of a 30 nucleobasecompound can be “fully complementary” to a target sequence that is 400nucleobases long. The 20 nucleobase portion of the 30 nucleobasecompound is fully complementary to the target sequence if the targetsequence has a corresponding 20 nucleobase portion wherein eachnucleobase is complementary to the 20 nucleobase portion of thecompound. At the same time, the entire 30 nucleobase compound may or maynot be fully complementary to the target sequence, depending on whetherthe remaining 10 nucleobases of the compound are also complementary tothe target sequence.

In certain embodiments, compounds described herein comprise one or moremismatched nucleobases relative to the target nucleic acid. In certainsuch embodiments, antisense activity against the target is reduced bysuch mismatch, but activity against a non-target is reduced by a greateramount. Thus, in certain such embodiments, selectivity of the compoundis improved. In certain embodiments, the mismatch is specificallypositioned within an oligonucleotide having a gapmer motif. In certainsuch embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8from the 5′-end of the gap region. In certain such embodiments, themismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3′-end of thegap region. In certain such embodiments, the mismatch is at position 1,2, 3, or 4 from the 5′-end of the wing region. In certain suchembodiments, the mismatch is at position 4, 3, 2, or 1 from the 3′-endof the wing region. In certain embodiments, the mismatch is specificallypositioned within an oligonucleotide not having a gapmer motif. Incertain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 from the 5′-end of the oligonucleotide. Incertain such embodiments, the mismatch is at position , 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 from the 3′-end of the oligonucleotide.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the compound. Alternatively, the non-complementary nucleobaseor nucleobases may be at an internal position of the compound. When twoor more non-complementary nucleobases are present, they may becontiguous (i.e. linked) or non-contiguous. In one embodiment, anon-complementary nucleobase is located in the wing segment of a gapmeroligonucleotide.

In certain embodiments, compounds described herein that are, or are upto 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in lengthcomprise no more than 4, no more than 3, no more than 2, or no more than1 non-complementary nucleobase(s) relative to a target nucleic acid,such as a PNPLA3 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein that are, or are upto 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleobases in length comprise no more than 6, no morethan 5, no more than 4, no more than 3, no more than 2, or no more than1 non-complementary nucleobase(s) relative to a target nucleic acid,such as a PNPLA3 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein also include thosewhich are complementary to a portion of a target nucleic acid. As usedherein, “portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof a compound. In certain embodiments, the compounds, are complementaryto at least an 8 nucleobase portion of a target segment. In certainembodiments, the compounds are complementary to at least a 9 nucleobaseportion of a target segment. In certain embodiments, the compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the compounds are complementary to at least an11 nucleobase portion of a target segment. In certain embodiments, thecompounds are complementary to at least a 12 nucleobase portion of atarget segment. In certain embodiments, the compounds are complementaryto at least a 13 nucleobase portion of a target segment. In certainembodiments, the compounds are complementary to at least a 14 nucleobaseportion of a target segment. In certain embodiments, the compounds arecomplementary to at least a 15 nucleobase portion of a target segment.In certain embodiments, the compounds are complementary to at least a 16nucleobase portion of a target segment. Also contemplated are compoundsthat are complementary to at least a 9, 10, 17, 18, 19, 20, or morenucleobase portion of a target segment, or a range defined by any two ofthese values.

Identity

The compounds provided herein may also have a defined percent identityto a particular nucleotide sequence, SEQ ID NO, or compound representedby a specific ION number, or portion thereof. In certain embodiments,compounds described herein are antisense compounds or oligomericcompounds. In certain embodiments, compounds described herein aremodified oligonucleotides. As used herein, a compound is identical tothe sequence disclosed herein if it has the same nucleobase pairingability. For example, a RNA which contains uracil in place of thymidinein a disclosed DNA sequence would be considered identical to the DNAsequence since both uracil and thymidine pair with adenine. Shortenedand lengthened versions of the compounds described herein as well ascompounds having non-identical bases relative to the compounds providedherein also are contemplated. The non-identical bases may be adjacent toeach other or dispersed throughout the compound. Percent identity of ancompound is calculated according to the number of bases that haveidentical base pairing relative to the sequence to which it is beingcompared.

In certain embodiments, compounds described herein, or portions thereof,are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to one or more of the compounds orSEQ ID NOs, or a portion thereof, disclosed herein. In certainembodiments, compounds described herein are about 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or anypercentage between such values, to a particular nucleotide sequence, SEQID NO, or compound represented by a specific ION number, or portionthereof, in which the compounds comprise an oligonucleotide having oneor more mismatched nucleobases. In certain such embodiments, themismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fromthe 5′-end ofthe oligonucleotide. In certain such embodiments, themismatch is at position , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the3′-end of the oligonucleotide.

In certain embodiments, compounds described herein comprise or consistof antisense compounds. In certain embodiments, a portion of theantisense compound is compared to an equal length portion of the targetnucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is comparedto an equal length portion of the target nucleic acid.

In certain embodiments, compounds described herein comprise or consistof oligonucleotides. In certain embodiments, a portion of theoligonucleotide is compared to an equal length portion of the targetnucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is comparedto an equal length portion of the target nucleic acid.

Certain Modified Compounds

In certain embodiments, compounds described herein comprise or consistof oligonucleotides consisting of linked nucleosides. Oligonucleotidesmay be unmodified oligonucleotides (RNA or DNA) or may be modifiedoligonucleotides. Modified oligonucleotides comprise at least onemodification relative to unmodified RNA or DNA (i.e., comprise at leastone modified nucleoside (comprising a modified sugar moiety and/or amodified nucleobase) and/or at least one modified internucleosidelinkage).

A. Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modifiednucleobase or both a modifed sugar moiety and a modified nucleobase.

1. Modified Sugar Moieties

In certain embodiments, sugar moieties are non-bicyclic modified sugarmoieties. In certain embodiments, modified sugar moieties are bicyclicor tricyclic sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of other types of modifiedsugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclicmodified furanosyl sugar moieties comprising one or more acyclicsubstituent, including, but not limited, to substituents at the 2′, 4′,and/or 5′ positions. In certain embodiments, the furanosyl sugar moietyis a ribosyl sugar moiety. In certain embodiments, one or more acyclicsubstituent of non-bicyclic modified sugar moieties is branched.Examples of 2′-substituent groups suitable for non-bicyclic modifiedsugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or“O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments,2′-substituent groups are selected from among: halo, allyl, amino,azido, SH, CN, OCN, CF₃, OCF₃, O-C₁-C₁₀ alkoxy, O-C₁-C₁₀ substitutedalkoxy, O-C₁-C₁₀ alkyl, O-C₁-C₁₀ substituted alkyl, S-alkyl,N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl,S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)) orOCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently,H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat.No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from among: hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen,alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groupssuitable for linearly non-bicyclic modified sugar moieties include, butare not limited to, alkoxy (e.g., methoxy), alkyl, and those describedin Manoharan et al., WO 2015/106128. Examples of 5′-substituent groupssuitable for non-bicyclic modified sugar moieties include, but are notlimited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certainembodiments, non-bicyclic modified sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties and the modified sugar moieties and modified nucleosidesdescribed in Migawa et al., WO 2008/101157 and Rajeev et al.,US2013/0203836.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂,CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O(CH₂)₂ON(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is,independently, H, an amino protecting group, or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃,O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andOCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

Nucleosides comprising modified sugar moieties, such as non-bicyclicmodified sugar moieties, are referred to by the position(s) of thesubstitution(s) on the sugar moiety of the nucleoside. For example,nucleosides comprising 2′-substituted or 2′-modified sugar moieties arereferred to as 2′-substituted nucleosides or 2′-modified nucleosides.

Certain modifed sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. In certain suchembodiments, the furanose ring is a ribose ring.

Examples of such 4′ to 2′ bridging sugar substituents include, but arenot limited to: 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′(“LNA”), 4′-CH₂—S-2′, 4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referredto as “constrained ethyl” or “cEt” when in the S configuration),4′-CH₂—O—CH₂-2′, 4′-CH₂—N(R)-2′, 4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE”or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. Pat. No.7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S.Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193),4′-C(CH₃)(CH₃)—O-2′ and analogs thereof (see, e.g., Seth et al., U.S.Pat. No. 8,278,283), 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g.,Prakash et al., U.S. Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g.,Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat.No. 8,124,745), 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org.Chem.,2009, 74, 118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (seee.g.,, Seth et al., U.S. Pat. No. 8,278,426),4′-C(R_(a)R_(b))—N(R)—O-2′, 4′-C(R_(a)R_(b))—O—N(R)-2′,4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′, wherein each R, R_(a), and R_(b)is, independently, H, a protecting group, or C₁-C₁₂ alkyl (see, e.g.Imanishi et al., U.S. Pat. No. 7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, forexample: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443,Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc., 2007, 129, 8362-8379; Elayadi et al., Curr. OpinionInvens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel etal., U.S. Pat. No. 7,053,207, Imanishi et al., U.S. Pat. No. 6,268,490,Imanishi et al. U.S. Pat. No. 6,770,748, Imanishi et al., U.S. RE44,779;Wengel et al., U.S. Pat. No. 6,794,499, Wengel et al., U.S. Pat. No.6,670,461; Wengel et al., U.S. Pat. No. 7,034,133, Wengel et al., U.S.Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel etal., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582;and Ramasamy et al., U.S. Pat. No. 6,525,191, Torsten et al., WO2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181;Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No.7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat.No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S.Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al.,U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth etal., U.S. Pat. No. 8,501,805; Allerson et al., US2008/0039618; andMigawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, an LNA nucleoside (describedherein) may be in the a-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or α-L-LNA bicyclic nucleosides have beenincorporated into oligonucleotides that showed antisense activity(Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein,general descriptions of bicyclic nucleosides include both isomericconfigurations. When the positions of specific bicyclic nucleosides(e.g., LNA or cEt) are identified in exemplified embodiments herein,they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the sugar moiety isreplaced, e.g., with a sulfur, carbon or nitrogen atom. In certain suchembodiments, such modified sugar moieties also comprise bridging and/ornon-bridging substituents as described herein. For example, certainsugar surrogates comprise a 4′-sulfur atom and a substitution at the2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat etal., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having otherthan 5 atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyransmay be further modified or substituted. Nucleosides comprising suchmodified tetrahydropyrans include, but are not limited to, hexitolnucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid(“MNA”) (see e.g., Leumann, C J. Bioorg. & Med. Chem. 2002,10, 841-854),fluoro HNA:

(“F-HNA”, see e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze etal., U.S. Pat. No. 8,440,803; and Swayze et al., U.S. Pat. No.9,005,906) F-HNA can also be referred to as a F-THP or 3′-fluorotetrahydropyran, and nucleosides comprising additional modified THPcompounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide or one of T₃ and T₄ is an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group, or a 5′ or 3′-terminal group; q₁, q₂,q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ isindependently selected from among: hydrogen, halogen, substituted orunsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂,NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided whereinq₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, atleast one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. Incertain embodiments, modified THP nucleosides are provided wherein oneof R₁ and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, incertain embodiments, R₁ is methoxy and R₂ is H, and in certainembodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example, nucleosidescomprising morpholino sugar moieties and their use in oligonucleotideshave been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41,4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton etal., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444;and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term“morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example, byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are refered to herein as “modifedmorpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieites.Examples of nucleosides and oligonucleotides comprising such acyclicsugar surrogates include, but are not limited to: peptide nucleic acid(“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org.Biomol. Chem., 2013, 11, 5853-5865), and nucleosides andoligonucleotides described in Manoharan et al., US2013/130378.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used in modified nucleosides.

2. Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.

In certain embodiments, compounds described herein comprise modifiedoligonucleotides. In certain embodiments, modified oligonucleotidescomprise one or more nucleoside comprising an unmodified nucleobase. Incertain embodiments, modified oligonucleotides comprise one or morenucleoside comprising a modified nucleobase. In certain embodiments,modified oligonucleotides comprise one or more nucleosides that does notcomprise a nucleobase, referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from:5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynylsubstituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6substituted purines. In certain embodiments, modified nucleobases areselected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine,5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine,6-N-methylguanine, 6-N-methyladenine, 2-propyladenine , 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-propynyl (C≡C—CH₃) uracil,5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine,5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo,particularly, 5-bromo, 5-trifluoromethyl, 5-halouracil, and5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine,2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine,4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine,5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases. Furthermodified nucleobases include tricyclic pyrimidines, such as1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one, and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example, 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in Merigan et al., U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859;Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and thosedisclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T.,Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above notedmodified nucleobases, as well as other modified nucleobases includewithout limitation, Manoharan et al., US2003/0158403, Manoharan et al.,US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al.,U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066;Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat.No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al.,U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cooket al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No.5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al.,U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No.5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S.Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook etal., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cooket al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903;Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No.5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al.,U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook etal., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No.6,005,096.

In certain embodiments, compounds targeted to a PNPLA3 nucleic acidcomprise one or more modified nucleobases. In certain embodiments, themodified nucleobase is 5-methylcytosine. In certain embodiments, eachcytosine is a 5-methylcytosine.

Modified Internucleoside Linkages

The naturally occuring internucleoside linkage of RNA and DNA is a 3′ to5′ phosphodiester linkage. In certain embodiments, compounds describedherein having one or more modified, i.e. non-naturally occurring,internucleoside linkages are often selected over compounds havingnaturally occurring internucleoside linkages because of desirableproperties such as, for example, enhanced cellular uptake, enhancedaffinity for target nucleic acids, and increased stability in thepresence of nucleases.

Representative internucleoside linkages having a chiral center includebut are not limited to alkylphosphonates and phosphorothioates. Modifiedoligonucleotides comprising internucleoside linkages having a chiralcenter can be prepared as populations of modified oligonucleotidescomprising stereorandom internucleoside linkages, or as populations ofmodified oligonucleotides comprising phosphorothioate linkages inparticular stereochemical configurations. In certain embodiments,populations of modified oligonucleotides comprise phosphorothioateinternucleoside linkages wherein all of the phosphorothioateinternucleoside linkages are stereorandom. Such modifiedoligonucleotides can be generated using synthetic methods that result inrandom selection of the stereochemical configuration of eachphosphorothioate linkage. Nonetheless, as is well understood by those ofskill in the art, each individual phosphorothioate of each individualoligonucleotide molecule has a defined stereoconfiguration. In certainembodiments, populations of modified oligonucleotides are enriched formodified oligonucleotides comprising one or more particularphosphorothioate internucleoside linkages in a particular, independentlyselected stereochemical configuration. In certain embodiments, theparticular configuration of the particular phosphorothioate linkage ispresent in at least 65% of the molecules in the population. In certainembodiments, the particular configuration of the particularphosphorothioate linkage is present in at least 70% of the molecules inthe population. In certain embodiments, the particular configuration ofthe particular phosphorothioate linkage is present in at least 80% ofthe molecules in the population. In certain embodiments, the particularconfiguration of the particular phosphorothioate linkage is present inat least 90% of the molecules in the population. In certain embodiments,the particular configuration of the particular phosphorothioate linkageis present in at least 99% of the molecules in the population. Suchchirally enriched populations of modified oligonucleotides can begenerated using synthetic methods known in the art, e.g., methodsdescribed in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid.Res. 42, 13456 (2014), and WO 2017/015555. In certain embodiments, apopulation of modified oligonucleotides is enriched for modifiedoligonucleotides having at least one indicated phosphorothioate in the(Sp) configuration. In certain embodiments, a population of modifiedoligonucleotides is enriched for modified oligonucleotides having atleast one phosphorothioate in the (Rp) configuration. In certainembodiments, modified oligonucleotides comprising (Rp) and/or (Sp)phosphorothioates comprise one or more of the following formulas,respectively, wherein “B” indicates a nucleobase:

Unless otherwise indicated, chiral internucleoside linkages of modifiedoligonucleotides described herein can be stereorandom or in a particularstereochemical configuration.

In certain embodiments, compounds targeted to a PNPLA3 nucleic acidcomprise one or more modified internucleoside linkages. In certainembodiments, the modified internucleoside linkages are phosphorothioatelinkages. In certain embodiments, each internucleoside linkage of anantisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, compounds described herein compriseoligonucleotides. Oligonucleotides having modified internucleosidelinkages include internucleoside linkages that retain a phosphorus atomas well as internucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, nucleosides of modified oligonucleotides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus-containinginternucleoside linkages include, but are not limited to, phosphates,which contain a phosphodiester bond (“P═O”) (also referred to asunmodified or naturally occurring linkages), phosphotriesters,methylphosphonates, phosphoramidates, and phosphorothioates (“P═S”), andphosphorodithioates (“HS—P═S”). Representative non-phosphorus containinginternucleoside linking groups include, but are not limited to,methylenemethylimino (—CH₂—N(CH₃)—O—CH₂), thiodiester, thionocarbamate(—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared tonaturally occurring phosphate linkages, can be used to alter, typicallyincrease, nuclease resistance of the oligonucleotide. In certainembodiments, internucleoside linkages having a chiral atom can beprepared as a racemic mixture, or as separate enantiomers.Representative chiral internucleoside linkages include, but are notlimited to, alkylphosphonates and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinginternucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), methoxypropyl, and thioformacetal (3′-S—CH₂—O-5′).Further neutral internucleoside linkages include nonionic linkagescomprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide,sulfide, sulfonate ester and amides (See, for example: CarbohydrateModifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds.,ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH₂ component parts.

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif. In such embodiments, the internucleoside linkages in eachof two wing regions are different from the internucleoside linkages inthe gap region. In certain embodiments, the internucleoside linkages inthe wings are phosphodiester and the internucleoside linkages in the gapare phosphorothioate. The nucleoside motif is independently selected, sosuch oligonucleotides having a gapped internucleoside linkage motif mayor may not have a gapped nucleoside motif and, if it does have a gappednucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides comprise a region of uniformly modified internucleosidelinkages. In certain such embodiments, the oligonucleotide comprises aregion that is uniformly linked by phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide is uniformlylinked by phosphorothioate. In certain embodiments, each internucleosidelinkage of the oligonucleotide is selected from phosphodiester andphosphorothioate. In certain embodiments, each internucleoside linkageof the oligonucleotide is selected from phosphodiester andphosphorothioate and at least one internucleoside linkage isphosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

In certain embodiments, oligonucleotides comprise one or moremethylphosphonate linkages. In certain embodiments, oligonucleotideshaving a gapmer nucleoside motif comprise a linkage motif comprising allphosphorothioate linkages except for one or two methylphosphonatelinkages. In certain embodiments, one methylphosphonate linkage is inthe central gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number ofphosphorothioate internucleoside linkages and phosphodiesterinternucleoside linkages to maintain nuclease resistance. In certainembodiments, it is desirable to arrange the number and position ofphosphorothioate internucleoside linkages and the number and position ofphosphodiester internucleoside linkages to maintain nuclease resistance.In certain embodiments, the number of phosphorothioate internucleosidelinkages may be decreased and the number of phosphodiesterinternucleoside linkages may be increased. In certain embodiments, thenumber of phosphorothioate internucleoside linkages may be decreased andthe number of phosphodiester internucleoside linkages may be increasedwhile still maintaining nuclease resistance. In certain embodiments, itis desirable to decrease the number of phosphorothioate internucleosidelinkages while retaining nuclease resistance. In certain embodiments, itis desirable to increase the number of phosphodiester internucleosidelinkages while retaining nuclease resistance.

3. Certain Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. Oligonucleotides can have a motif, e.g. a pattern ofunmodified and/or modified sugar moieties, nucleobases, and/orinternucleoside linkages. In certain embodiments, modifiedoligonucleotides comprise one or more modified nucleosides comprising amodified sugar. In certain embodiments, modified oligonucleotidescomprise one or more modified nucleosides comprising a modifiednucleobase. In certain embodiments, modified oligonucleotides compriseone or more modified internucleoside linkage. In such embodiments, themodified, unmodified, and differently modified sugar moieties,nucleobases, and/or internucleoside linkages of a modifiedoligonucleotide define a pattern or motif. In certain embodiments, thepatterns of sugar moieties, nucleobases, and internucleoside linkagesare each independent of one another. Thus, a modified oligonucleotidemay be described by its sugar motif, nucleobase motif and/orinternucleoside linkage motif (as used herein, nucleobase motifdescribes the modifications to the nucleobases independent of thesequence of nucleobases).

a. Certain Sugar Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprise oneor more type of modified sugar and/or unmodified sugar moiety arrangedalong the oligonucleotide or region thereof in a defined pattern orsugar motif. In certain instances, such sugar motifs include, but arenot limited to, any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist ofa region having a gapmer motif, which comprises two external regions or“wings” and a central or internal region or “gap”. The three regions ofa gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguoussequence of nucleosides, wherein at least some of the sugar moieties ofthe nucleosides of each of the wings differ from at least some of thesugar moieties of the nucleosides of the gap. Specifically, at least thesugar moieties of the nucleosides of each wing that are closest to thegap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside ofthe 3′-wing) differ from the sugar moiety of the neighboring gapnucleosides, thus defining the boundary between the wings and the gap(i.e., the wing/gap junction). In certain embodiments, the sugarmoieties within the gap are the same as one another. In certainembodiments, the gap includes one or more nucleosides having a sugarmoiety that differs from the sugar moiety of one or more othernucleosides of the gap. In certain embodiments, the sugar motifs of thetwo wings are the same as one another (symmetric gapmer). In certainembodiments, the sugar motif of the 5′-wing differs from the sugar motifof the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides.In certain embodiments, the nucleosides of a gapmer are all modifiednucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides.In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 10 nucleosides. Incertain embodiments, each nucleoside of the gap of a gapmer is anunmodified 2′-deoxy nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In suchembodiments, the nucleosides on the gap side of each wing/gap junctionare unmodified 2′-deoxy nucleosides and the nucleosides on the wingsides of each wing/gap junction are modified nucleosides. In certainsuch embodiments, each nucleoside of the gap is an unmodified 2′-deoxynucleoside. In certain such embodiments, each nucleoside of each wing isa modified nucleoside.

In certain embodiments, a modified oligonucleotide has a fully modifiedsugar motif wherein each nucleoside of the modified oligonucleotidecomprises a modified sugar moiety. In certain embodiments, modifiedoligonucleotides comprise or consist of a region having a fully modifiedsugar motif wherein each nucleoside of the region comprises a modifiedsugar moiety. In certain embodiments, modified oligonucleotides compriseor consist of a region having a fully modified sugar motif, wherein eachnucleoside within the fully modified region comprises the same modifiedsugar moiety, referred to herein as a uniformly modified sugar motif. Incertain embodiments, a fully modified oligonucleotide is a uniformlymodified oligonucleotide. In certain embodiments, each nucleoside of auniformly modified comprises the same 2′-modification.

b. Certain Nucleobase Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprisemodified and/or unmodified nucleobases arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, each nucleobase is modified. In certainembodiments, none of the nucleobases are modified. In certainembodiments, each purine or each pyrimidine is modified. In certainembodiments, each adenine is modified. In certain embodiments, eachguanine is modified. In certain embodiments, each thymine is modified.In certain embodiments, each uracil is modified. In certain embodiments,each cytosine is modified. In certain embodiments, some or all of thecytosine nucleobases in a modified oligonucleotide are5-methylcytosines.

In certain embodiments, modified oligonucleotides comprise a block ofmodified nucleobases. In certain such embodiments, the block is at the3′-end of the oligonucleotide. In certain embodiments, the block iswithin 3 nucleosides of the 3′-end of the oligonucleotide. In certainembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments, the block is within 3 nucleosides of the 5′-end ofthe oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprisea nucleoside comprising a modified nucleobase. In certain suchembodiments, one nucleoside comprising a modified nucleobase is in thecentral gap of an oligonucleotide having a gapmer motif. In certain suchembodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosylmoiety. In certain embodiments, the modified nucleobase is selectedfrom: a 2-thiopyrimidine and a 5-propynepyrimidine.

c. Certain Internucleoside Linkage Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprisemodified and/or unmodified internucleoside linkages arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, essentially each internucleoside linking group is aphosphate internucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of a modified oligonucleotide is aphosphorothioate (P═S). In certain embodiments, each internucleosidelinking group of a modified oligonucleotide is independently selectedfrom a phosphorothioate and phosphate internucleoside linkage. Incertain embodiments, the sugar motif of a modified oligonucleotide is agapmer and the internucleoside linkages within the gap are all modified.In certain such embodiments, some or all of the internucleoside linkagesin the wings are unmodified phosphate linkages. In certain embodiments,the terminal internucleoside linkages are modified. In certainembodiments, the sugar motif of a modified oligonucleotide is a gapmer,and the internucleoside linkage motif comprises at least onephosphodiester internucleoside linkage in at least one wing, wherein theat least one phosphodiester linkage is not a terminal internucleosidelinkage, and the remaining internucleoside linkages are phosphorothioateinternucleoside linkages. In certain such embodiments, all of thephosphorothioate linkages are stereorandom. In certain embodiments, allof the phosphorothioate linkages in the wings are (Sp)phosphorothioates, and the gap comprises at least one Sp, Sp, Rp motif.In certain embodiments, populations of modified oligonucleotides areenriched for modified oligonucleotides comprising such internucleosidelinkage motifs.

4. Certain Modified Oligonucleotides

In certain embodiments, compounds described herein comprise modifiedoligonucleotides. In certain embodiments, the above modifications(sugar, nucleobase, internucleoside linkage) are incorporated into amodified oligonucleotide. In certain embodiments, modifiedoligonucleotides are characterized by their modification, motifs, andoverall lengths. In certain embodiments, such parameters are eachindependent of one another. Thus, unless otherwise indicated, eachinternucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. For example, theinternucleoside linkages within the wing regions of a sugar gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region of thesugar motif. Likewise, such gapmer oligonucleotides may comprise one ormore modified nucleobases independent of the gapmer pattern of the sugarmodifications. Furthermore, in certain instances, an oligonucleotide isdescribed by an overall length or range and by lengths or length rangesof two or more regions (e.g., a regions of nucleosides having specifiedsugar modifications). In such circumstances, it may be possible toselect numbers for each range that result in an oligonucleotide havingan overall length falling outside the specified range. In suchcircumstances, both elements must be satisfied. For example, in certainembodiments, a modified oligonucleotide consists of 15-20 linkednucleosides and has a sugar motif consisting of three regions, A, B, andC, wherein region A consists of 2-6 linked nucleosides having aspecified sugar motif, region B consists of 6-10 linked nucleosideshaving a specified sugar motif, and region C consists of 2-6 linkednucleosides having a specified sugar motif. Such embodiments do notinclude modified oligonucleotides where A and C each consist of 6 linkednucleosides and B consists of 10 linked nucleosides (even though thosenumbers of nucleosides are permitted within the requirements for A, B,and C) because the overall length of such oligonucleotide will be 22,which exceeds the upper limit of the overall length of the modifiedoligonucleotide (20). Herein, if a description of an oligonucleotide issilent with respect to one or more parameters, such parameter is notlimited. Thus, a modified oligonucleotide described only as having agapmer sugar motif without further description may have any length,internucleoside linkage motif, and nucleobase motif. Unless otherwiseindicated, all modifications are independent of nucleobase sequence.

Certain Conjugated Compounds

In certain embodiments, the compounds described herein comprise orconsist of an oligonucleotide (modified or unmodified) and, optionally,one or more conjugate groups and/or terminal groups. Conjugate groupsconsist of one or more conjugate moiety and a conjugate linker whichlinks the conjugate moiety to the oligonucleotide. Conjugate groups maybe attached to either or both ends of an oligonucleotide and/or at anyinternal position. In certain embodiments, conjugate groups are attachedto the 2′-position of a nucleoside of a modified oligonucleotide. Incertain embodiments, conjugate groups that are attached to either orboth ends of an oligonucleotide are terminal groups. In certain suchembodiments, conjugate groups or terminal groups are attached at the 3′and/or 5′-end of oligonucleotides. In certain such embodiments,conjugate groups (or terminal groups) are attached at the 3′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 3′-end of oligonucleotides. In certain embodiments, conjugategroups (or terminal groups) are attached at the 5′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 5′-end of oligonucleotides.

In certain embodiments, the oligonucleotide is modified. In certainembodiments, the oligonucleotide of a compound has a nucleobase sequencethat is complementary to a target nucleic acid. In certain embodiments,oligonucleotides are complementary to a messenger RNA (mRNA). In certainembodiments, oligonucleotides are complementary to a pre-mRNA. Incertain embodiments, oligonucleotides are complementary to a sensetranscript.

Examples of terminal groups include but are not limited to conjugategroups, capping groups, phosphate moieties, protecting groups, modifiedor unmodified nucleosides, and two or more nucleosides that areindependently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to oneor more conjugate groups. In certain embodiments, conjugate groupsmodify one or more properties of the attached oligonucleotide,including, but not limited to, pharmacodynamics, pharmacokinetics,stability, binding, absorption, tissue distribution, cellulardistribution, cellular uptake, charge and clearance. In certainembodiments, conjugate groups impart a new property on the attachedoligonucleotide, e.g., fluorophores or reporter groups that enabledetection of the oligonucleotide.

Certain conjugate groups and conjugate moieties have been describedpreviously, for example: cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3,2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanovet al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic, a palmityl moiety (Mishra et al.,Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, i, 923-937), a tocopherol group (Nishina et al.,Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038/mtna.2014.72and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAccluster (e.g., WO2014/179620).

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reportermolecules, polyamines, polyamides, peptides, carbohydrates (e.g.,GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers,cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores,and dyes.

In certain embodiments, a conjugate moiety comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial, or an antibiotic.

2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugatelinkers. In certain embodiments, a conjugate group is a single chemicalbond (i.e. conjugate moiety is attached to an oligonucleotide via aconjugate linker through a single bond). In certain embodiments, theconjugate linker comprises a chain structure, such as a hydrocarbylchain, or an oligomer of repeating units, such as ethylene glycol,nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groupsselected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol,ether, thioether, and hydroxylamino. In certain such embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus moiety. In certain embodiments, the conjugate linkercomprises at least one phosphate group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugatelinkers described above, are bifunctional linking moieties, e.g., thoseknown in the art to be useful for attaching conjugate groups to parentcompounds, such as the oligonucleotides provided herein. In general, abifunctional linking moiety comprises at least two functional groups.One of the functional groups is selected to bind to a particular site ona compound and the other is selected to bind to a conjugate group.Examples of functional groups used in a bifunctional linking moietyinclude, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In certain embodiments, bifunctional linking moieties compriseone or more groups selected from amino, hydroxyl, carboxylic acid,thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include, but are not limited to,pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, butare not limited to, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, andalkynyl.

In certain embodiments, conjugate linkers comprise 1-10linker-nucleosides. In certain embodiments, such linker-nucleosides aremodified nucleosides. In certain embodiments, such linker-nucleosidescomprise a modified sugar moiety. In certain embodiments,linker-nucleosides are unmodified. In certain embodiments,linker-nucleosides comprise an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, a cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typicallydesirable for linker-nucleosides to be cleaved from the compound afterit reaches a target tissue. Accordingly, linker-nucleosides aretypically linked to one another and to the remainder of the compoundthrough cleavable bonds. In certain embodiments, such cleavable bondsare phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of theoligonucleotide. Accordingly, in embodiments in which a compoundcomprises an oligonucleotide consisting of a specified number or rangeof linked nucleosides and/or a specified percent complementarity to areference nucleic acid and the compound also comprises a conjugate groupcomprising a conjugate linker comprising linker-nucleosides, thoselinker-nucleosides are not counted toward the length of theoligonucleotide and are not used in determining the percentcomplementarity of the oligonucleotide for the reference nucleic acid.For example, a compound may comprise (1) a modified oligonucleotideconsisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10linker-nucleosides that are contiguous with the nucleosides of themodified oligonucleotide. The total number of contiguous linkednucleosides in such a compound is more than 30. Alternatively, acompound may comprise a modified oligonucleotide consisting of 8-30nucleosides and no conjugate group. The total number of contiguouslinked nucleosides in such a compound is no more than 30. Unlessotherwise indicated, conjugate linkers comprise no more than 10linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 5 linker-nucleosides. In certain embodiments, conjugatelinkers comprise no more than 3 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 2linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 1 linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to becleaved from the oligonucleotide. For example, in certain circumstances,compounds comprising a particular conjugate moiety are better taken upby a particular cell type, but once the compound has been taken up, itis desirable that the conjugate group be cleaved to release theunconjugated or parent oligonucleotide. Thus, certain conjugates maycomprise one or more cleavable moieties, typically within the conjugatelinker. In certain embodiments, a cleavable moiety is a cleavable bond.In certain embodiments, a cleavable moiety is a group of atomscomprising at least one cleavable bond. In certain embodiments, acleavable moiety comprises a group of atoms having one, two, three,four, or more than four cleavable bonds. In certain embodiments, acleavable moiety is selectively cleaved inside a cell or subcellularcompartment, such as a lysosome. In certain embodiments, a cleavablemoiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: anamide, an ester, an ether, one or both esters of a phosphodiester, aphosphate ester, a carbamate, or a disulfide. In certain embodiments, acleavable bond is one or both of the esters of a phosphodiester. Incertain embodiments, a cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is aphosphate linkage between an oligonucleotide and a conjugate moiety orconjugate group.

In certain embodiments, a cleavable moiety comprises or consists of oneor more linker-nucleosides. In certain such embodiments, one or morelinker-nucleosides are linked to one another and/or to the remainder ofthe compound through cleavable bonds. In certain embodiments, suchcleavable bonds are unmodified phosphodiester bonds. In certainembodiments, a cleavable moiety is 2′-deoxy nucleoside that is attachedto either the 3′ or 5′-terminal nucleoside of an oligonucleotide by aphosphate internucleoside linkage and covalently attached to theremainder of the conjugate linker or conjugate moiety by a phosphate orphosphorothioate linkage. In certain such embodiments, the cleavablemoiety is 2′-deoxyadenosine.

3. Certain Cell-Targeting Conjugate Moieties

In certain embodiments, a conjugate group comprises a cell-targetingconjugate moiety. In certain embodiments, a conjugate group has thegeneral formula:

wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certainembodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1,j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. Incertain embodiments, n is 2, j is 0 and k is 1. In certain embodiments,n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and kis 0. In certain embodiments, n is 3, j is 0 and k is 1. In certainembodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targetingmoieties that have at least one tethered ligand. In certain embodiments,cell-targeting moieties comprise two tethered ligands covalentlyattached to a branching group. In certain embodiments, cell-targetingmoieties comprise three tethered ligands covalently attached to abranching group.

In certain embodiments, the cell-targeting moiety comprises a branchinggroup comprising one or more groups selected from alkyl, amino, oxo,amide, disulfide, polyethylene glycol, ether, thioether, andhydroxylamino groups. In certain embodiments, the branching groupcomprises a branched aliphatic group comprising groups selected fromalkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether,thioether, and hydroxylamino groups. In certain such embodiments, thebranched aliphatic group comprises groups selected from alkyl, amino,oxo, amide, and ether groups. In certain such embodiments, the branchedaliphatic group comprises groups selected from alkyl, amino, and ethergroups. In certain such embodiments, the branched aliphatic groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem.

In certain embodiments, each tether of a cell-targeting moiety comprisesone or more groups selected from alkyl, substituted alkyl, ether,thioether, disulfide, amino, oxo, amide, phosphodiester, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether, thioether, disulfide, amino, oxo, amide, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, phosphodiester, ether, amino, oxo, and amide, inany combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl,ether, amino, oxo, and amide, in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl, amino, and oxo, in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl and oxo, in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl and phosphodiester, in anycombination. In certain embodiments, each tether comprises at least onephosphorus linking group or neutral linking group. In certainembodiments, each tether comprises a chain from about 6 to about 20atoms in length. In certain embodiments, each tether comprises a chainfrom about 10 to about 18 atoms in length. In certain embodiments, eachtether comprises about 10 atoms in chain length.

In certain embodiments, each ligand of a cell-targeting moiety has anaffinity for at least one type of receptor on a target cell. In certainembodiments, each ligand has an affinity for at least one type ofreceptor on the surface of a mammalian liver cell. In certainembodiments, each ligand has an affinity for the hepaticasialoglycoprotein receptor (ASGP-R). In certain embodiments, eachligand is a carbohydrate. In certain embodiments, each ligand is,independently selected from galactose, N-acetyl galactoseamine (GalNAc),mannose, glucose, glucoseamine, and fucose. In certain embodiments, eachligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, thecell-targeting moiety comprises 3 GalNAc ligands. In certainembodiments, the cell-targeting moiety comprises 2 GalNAc ligands. Incertain embodiments, the cell-targeting moiety comprises 1 GalNAcligand.

In certain embodiments, each ligand of a cell-targeting moiety is acarbohydrate, carbohydrate derivative, modified carbohydrate,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain such embodiments, the conjugate group comprises acarbohydrate cluster (see, e.g., Maier et al., “Synthesis of AntisenseOligonucleotides Conjugated to a Multivalent Carbohydrate Cluster forCellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensenet al., “Design and Synthesis of Novel N-Acetylgalactosamine-TerminatedGlycolipids for Targeting of Lipoproteins to the HepaticAsiaglycoprotein Receptor,” J. Med. Chem. 2004, 47, 5798-5808, which areincorporated herein by reference in their entirety). In certain suchembodiments, each ligand is an amino sugar or a thio sugar. For example,amino sugars may be selected from any number of compounds known in theart, such as sialic acid, α-D-galactosamine, β-muramic acid,2-deoxy-2-methylamino-L-glucopyranose,4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom 5-Thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, compounds described herein comprise a conjugategroup described herein as

“LICA-1”. LICA-1 is shown below without the optional cleavable moiety atthe end of the conjugate linker:

In certain embodiments, compounds described herein comprise LICA-1 and acleavable moiety within the conjugate linker have the formula:

wherein ‘oligo’ is an oligonucleotide.

Representative publications that teach the preparation of certain of theabove noted conjugate groups and compounds comprising conjugate groups,tethers, conjugate linkers, branching groups, ligands, cleavablemoieties as well as other modifications include, without limitation,U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177,7,491,805, 8,106,022, 7,723,509, 9,127,276, US 2006/0148740, US2011/0123520, WO 2013/033230 and WO 2012/037254, Biessen et al., J. Med.Chem. 1995, 38, 1846-1852, Lee et al., Bioorganic & Medicinal Chemistry2011,19, 2494-2500, Rensen et al., J. Biol. Chem. 2001, 276,37577-37584, Rensen et al., J. Med. Chem. 2004, 47, 5798-5808, Sliedregtet al., J. Med. Chem. 1999, 42, 609-618, and Valentijn et al.,Tetrahedron, 1997, 53, 759-770, each of which is incorporated byreference herein in its entirety.

In certain embodiments, compounds described herein comprise modifiedoligonucleotides comprising a gapmer or fully modified motif and aconjugate group comprising at least one, two, or three GalNAc ligands.In certain embodiments, compounds described herein comprise a conjugategroup found in any of the following references: Lee, Carbohydr Res,1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945;Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al.,Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4,317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessenet al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron,1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490;Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol,2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584;Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al.,Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006,16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15,7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee etal., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

Compositions and Methods for Formulating Pharmaceutical Compositions

Compounds described herein may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Certain embodiments provide pharmaceutical compositions comprising oneor more compounds or a salt thereof. In certain embodiments, thecompounds are antisense compounds or oligomeric compounds. In certainembodiments, the compounds comprise or consist of a modifiedoligonucleotide. In certain such embodiments, the pharmaceuticalcomposition comprises a suitable pharmaceutically acceptable diluent orcarrier. In certain embodiments, a pharmaceutical composition comprisesa sterile saline solution and one or more compound. In certainembodiments, such pharmaceutical composition consists of a sterilesaline solution and one or more compound. In certain embodiments, thesterile saline is pharmaceutical grade saline. In certain embodiments, apharmaceutical composition comprises one or more compound and sterilewater. In certain embodiments, a pharmaceutical composition consists ofone compound and sterile water. In certain embodiments, the sterilewater is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more compounds andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more compound and sterilePBS. In certain embodiments, the sterile PBS is pharmaceutical gradePBS. Compositions and methods for the formulation of pharmaceuticalcompositions are dependent upon a number of criteria, including, but notlimited to, route of administration, extent of disease, or dose to beadministered.

A compound described herein targeted to PNPLA3 nucleic acid can beutilized in pharmaceutical compositions by combining the compound with asuitable pharmaceutically acceptable diluent or carrier. In certainembodiments, a pharmaceutically acceptable diluent is water, such assterile water suitable for injection. Accordingly, in one embodiment,employed in the methods described herein is a pharmaceutical compositioncomprising a compound targeted to PNPLA3 nucleic acid and apharmaceutically acceptable diluent. In certain embodiments, thepharmaceutically acceptable diluent is water. In certain embodiments,the compound comprises or consists of a modified oligonucleotideprovided herein.

Pharmaceutical compositions comprising compounds provided hereinencompass any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other oligonucleotide which, upon administration toan animal, including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof Incertain embodiments, the compounds are antisense compounds or oligomericcompounds. In certain embodiments, the compound comprises or consists ofa modified oligonucleotide. Accordingly, for example, the disclosure isalso drawn to pharmaceutically acceptable salts of compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of a compound which are cleaved by endogenous nucleaseswithin the body, to form the active compound.

In certain embodiments, the compounds or compositions further comprise apharmaceutically acceptable carrier or diluent.

Certain Selected Compounds

Approximately 2,384 newly designed compounds of various lengths,chemistries, and motifs were tested for their effect on human PNPLA3mRNA in vitro in several cell types (Example 1). Of 2,384 compoundstested for potency at a single dose in vitro, over 400 selectedcompounds were tested for dose dependent inhibition in A431 cells(Example 2). Of the over 400 compounds tested by dose response assays,the compounds were further screened for high dose tolerability in aBALB/c mouse model and 87 oligonucleotides were selected for in vivoefficacy in a PNPLA3 transgenic mouse model.

Of the 87 oligonucleotides tested in the transgenic mouse model, 23oligonucleotides were selected to be further tested for tolerability inpreclinical rodel models. In the in vivo rodent tolerability models,body weights and organ weights, liver function markers (such as alaninetransaminase, aspartate transaminase and bilirubin), and kidney functionmarkers (such as BUN and creatinine) were measured. In the CD1 mousemodel and in the Sprague-Dawley rat model, ION 975591, 975605, 975612,975613, 975616, 975617, 975735, 975736, 994282, and 994284 were foundtolerable (Examples 5 and 6).

These compounds were further tested for efficacy in multi-dose assays inPNPLA3 transgenic mice (Example 7).

IONs 994284, 97605, 975616, 994282, 975613, 975617, 975735, 975736, and975612 were tested for tolerability in cynomolgus monkeys (Example 8).Treatment with the compounds was well tolerated in the monkeys.

Accordingly, provided herein are compounds with any one or more of theimproved properties. In certain embodiments, the compounds as describedherein are potent and tolerable.

EXAMPLES

The Examples below describe the screening process to identify leadcompounds targeted to PNPLA3. ION 994284, 97605, 975616, 994282, 975613,975617, 975735, 975736, and 975612 resulted in high potency andtolerability.

Non-Limiting Disclosure and Incorporation by Reference

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to, those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified

RNA and/or DNA, including, but not limited to, such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotides having such nucleobase sequence, whether modifiedor unmodified, including, but not limited to, such compounds comprisingRNA bases, such as those having sequence “AUCGAUCG” and those havingsome DNA bases and some RNA bases such as “AUCGATCG” and compoundshaving other modified nucleobases, such as “AT^(m)CGAUCG,” wherein ^(m)Cindicates a cytosine base comprising a methyl group at the 5-position.

Certain compounds described herein (e.g. modified oligonucleotides) haveone or more asymmetric centers and thus give rise to enantiomers,diastereomers, and other stereoisomeric configurations that may bedefined, in terms of absolute stereochemistry, as (R) or (S), as α or β,such as for sugar anomers, or as (D) or (L), such as for amino acids,etc. Compounds provided herein that are drawn or described as havingcertain stereoisomeric configurations include only the indicatedcompounds. Compounds provided herein that are drawn or described withundefined stereochemistry include all such possible isomers, includingtheir stereorandom and optically pure forms. Likewise, all tautomericforms of the compounds provided herein are included unless otherwiseindicated. Unless otherwise indicated, oligomeric compounds and modifiedoligonucleotides described herein are intended to include correspondingsalt forms.

Compounds described herein include variations in which one or more atomsare replaced with a non-radioactive isotope or radioactive isotope ofthe indicated element. For example, compounds herein that comprisehydrogen atoms encompass all possible deuterium substitutions for eachof the ¹H hydrogen atoms. Isotopic substitutions encompassed by thecompounds herein include, but are not limited to: ²H or ³H in place of¹H, ¹³C or ¹⁴C in place of ¹²C, ¹⁵N in place of ¹⁴N, ¹⁷O or ¹⁸O in placeof ¹⁶O, and ³³S, ³⁴S, ³⁵S, or ³⁶S in place of ³²S.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of Human PNPLA3 in A431 Cells

Antisense oligonucleotides were designed targeting a PNPLA3 nucleic acidand were tested for their effects on PNPLA3 mRNA in vitro. The antisenseoligonucleotides were tested in a series of experiments that had similarculture conditions. The results for each experiment are presented inseparate tables shown below.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as 3-10-3 cEt gapmers. The gapmers are 16nucleosides in length, wherein the central gap segment comprises of ten2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising three nucleosides. Each nucleoside inthe 5′ wing segment and each nucleoside in the 3′ wing segment has a cEtsugar modification. The internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytosine residues throughouteach gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the gapmer is targeted human gene sequence. Eachgapmer listed in the Tables below is targeted to either the human PNPLA3mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No.NM_025225.2) or the human PNPLA3 genomic sequence, designated herein asSEQ ID NO: 2 (GENBANK Accession No. NC_000022.11 truncated fromnucleotides 43921001 to 43954500). ‘n/a’ indicates that the antisenseoligonucleotide does not target that particular gene sequence with 100%complementarity.

Study 1

Cultured A431 cells at a density of 20,000 cells per well weretransfected by free uptake with 4,000 nM antisense oligonucleotide.After a treatment period of approximately 24 hours, RNA was isolatedfrom the cells and PNPLA3 mRNA levels were measured by quantitativereal-time PCR. Human primer probe set RTS36070 (forward sequenceCCTTGGTATGTTCCTGCTTCA, designated herein as SEQ ID NO: 11; reversesequence GTTGTCACTCACTCCTCCATC, designated herein as SEQ ID NO: 12;probe sequence TGGCCTTATCCCTCCTTCCTTCAGA, designated herein as SEQ IDNO: 13) was used to measure mRNA levels. PNPLA3 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of PNPLA3, relative tountreated control cells.

TABLE 1 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 912709 27 42 2765 2780GGCATTCCCAGCGCGA 0 17 912710 95 110 2833 2848 TCCTGATCCGCAGCAG 15 18912711 103 118 2841 2856 GGCTCGGGTCCTGATC 0 19 912712 131 146 2869 2884GTTAGGATCTGGGTCG 91 20 912713 164 179 2902 2917 GTACATGGCGGCGGCG 0 21912714 183 198 2921 2936 TCCAGCCGCGCTCTGC 23 22 912715 196 211 2934 2949GCGAAGGACAAGCTCC 60 23 912716 197 212 2935 2950 CGCGAAGGACAAGCTC 0 24912717 272 287 3010 3025 GCGGAGGAGGTGCGGG 0 25 912718 273 288 3011 3026CGCGGAGGAGGTGCGG 0 26 912719 274 289 3012 3027 TCGCGGAGGAGGTGCG 19 27912720 290 305 3028 3043 GAACAACATGCGCGCG 0 28 912721 291 306 3029 3044CGAACAACATGCGCGC 7 29 912722 292 307 3030 3045 CCGAACAACATGCGCG 21 30912723 293 308 3031 3046 GCCGAACAACATGCGC 0 31 912724 294 309 3032 3047CGCCGAACAACATGCG 0 32 912725 323 338 3061 3076 GCCGACGCAGTGCAAC 0 33912726 324 339 3062 3077 CGCCGACGCAGTGCAA 0 34 912727 340 355 3078 3093GGGATACCGGAGAGGA 43 35 912728 370 385 5944 5959 TCTGAGAGGACCTGCA 53 36912729 375 390 5949 5964 CAAGATCTGAGAGGAC 64 37 912730 404 419 5978 5993GCCAATGTTCCGACTC 71 38 912731 410 425 5984 5999 GAAGATGCCAATGTTC 51 39912732 429 444 6003 6018 TTAAGTTGAAGGATGG 96 40 912733 432 447 6006 6021TGCTTAAGTTGAAGGA 90 41 912734 478 493 6052 6067 TGGACATTGGCCGGGA 85 42912735 479 494 6053 6068 GTGGACATTGGCCGGG 50 43 912736 484 499 6058 6073AGCTGGTGGACATTGG 64 44 912737 528 543 6102 6117 CATCAGACACTCTGGT 5 45912738 531 546 6105 6120 CCCCATCAGACACTCT 73 46 912739 552 567 6126 6141AGTCAGACACCAGAAC 54 47 912755 693 708 11911 11926 TGGCATCAATGAAGGG 74 48912756 698 713 11916 11931 TGTTTTGGCATCAATG 91 49 912757 746 761 1196411979 TTTAGGGCAGATGTCG 89 50 912758 747 762 11965 11980 CTTTAGGGCAGATGTC90 51 912759 795 810 12013 12028 GTAGACTGAGCTTGGT 98 52 912760 820 83512038 12053 AGGTAGAGGTTCCCTG 0 53 912761 841 856 12059 12074GGGACAAAAGCTCTCG 20 54 912762 873 888 13609 13624 GGCATATCTCTCCCAG 0 55912763 874 889 13610 13625 AGGCATATCTCTCCCA 0 56 912764 886 901 1362213637 AAATATCCTCGAAGGC 57 57 912765 888 903 13624 13639 CCAAATATCCTCGAAG30 58 912766 889 904 13625 13640 TCCAAATATCCTCGAA 38 59 912767 894 90913630 13645 ATGCATCCAAATATCC 58 60 912768 925 940 N/A N/ATTGCAGATGCCCTTCT 15 61 912769 968 983 16088 16103 ATCCATCCCTTCTGAG 34 62912770 986 1001 16106 16121 GGGCATGGCGACCTCA 0 63 912771 1004 1019 1612416139 ACTCATGTTTGCCCAG 67 64 912772 1068 1083 16188 16203GGTCTAGCAGCTCATC 89 65 912773 1075 1090 16195 16210 CGCAGGTGGTCTAGCA 066 912774 1076 1091 16196 16211 ACGCAGGTGGTCTAGC 25 67 912775 1080 109516200 16215 TGAGACGCAGGTGGTC 50 68 912776 1086 1101 16206 16221GGATGCTGAGACGCAG 67 69 912777 1172 1187 19012 19027 GTATCCACCTTTGTCT 7870 912778 1178 1193 19018 19033 GCTCATGTATCCACCT 79 71 912779 1187 120219027 19042 GCAAATCTTGCTCATG 3 72 912780 1188 1203 19028 19043TGCAAATCTTGCTCAT 13 73 912781 1189 1204 19029 19044 TTGCAAATCTTGCTCA 074 912782 1195 1210 19035 19050 AGCAAGTTGCAAATCT 77 75 912783 1199 121419039 19054 GGGTAGCAAGTTGCAA 74 76 912784 1205 1220 19045 19060CCTAATGGGTAGCAAG 62 77 912785 1206 1221 19046 19061 TCCTAATGGGTAGCAA 7978

TABLE 2 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 912786 1207 1222 19047 19062ATCCTAATGGGTAGCA 81 79 912787 1211 1226 19051 19066 CATTATCCTAATGGGT 4680 912788 1212 1227 19052 19067 ACATTATCCTAATGGG 0 81 912789 1213 122819053 19068 GACATTATCCTAATGG 70 82 912790 1220 1235 19060 19075TACATAAGACATTATC 34 83 912791 1224 1239 19064 19079 GCATTACATAAGACAT 8684 912792 1245 1260 19085 19100 CCACAGGCAGGGTACA 76 85 912793 1246 126119086 19101 TCCACAGGCAGGGTAC 28 86 912794 1253 1268 19093 19108GGCAGATTCCACAGGC 75 87 912795 1259 1274 19099 19114 CGCAATGGCAGATTCC 9288 912796 1265 1280 19105 19120 GACAATCGCAATGGCA 64 89 912797 1266 128119106 19121 GGACAATCGCAATGGC 75 90 912798 1267 1282 19107 19122TGGACAATCGCAATGG 73 91 912799 1285 1300 23690 23705 AGCCATGTCACCAGTC 6792 912800 1289 1304 23694 23709 TGGAAGCCATGTCACC 24 93 912801 1290 130523695 23710 CTGGAAGCCATGTCAC 72 94 912802 1297 1312 23702 23717GGCATATCTGGAAGCC 0 95 912803 1298 1313 23703 23718 GGGCATATCTGGAAGC 0 96912804 1351 1366 23756 23771 AGCACTCGAGTGAACA 0 97 912805 1386 1401 N/AN/A GCATTTGGGACCTGGA 77 98 912806 1387 1402 N/A N/A GGCATTTGGGACCTGG 6099 912807 1388 1403 25151 25166 TGGCATTTGGGACCTG 41 100 912808 1394 140925157 25172 GCTCACTGGCATTTGG 44 101 912809 1523 1538 25286 25301GTTCAGGCTGGACCTG 49 102 912810 1547 1562 25310 25325 AGGTACTTTATTGCCC 11103 912811 1550 1565 25313 25328 AGCAGGTACTTTATTG 64 104 912812 16531668 25416 25431 AACTTTAGCACCTCTG 91 105 912813 1655 1670 25418 25433GAAACTTTAGCACCTC 88 106 912814 1656 1671 25419 25434 GGAAACTTTAGCACCT 53107 912815 1669 1684 25432 25447 CTGCACAAAGATGGGA 80 108 912816 16711686 25434 25449 AGCTGCACAAAGATGG 45 109 912817 1685 1700 25448 25463AGCAATGCGGAGGTAG 15 110 912818 1740 1755 25503 25518 ACCAACTCAGCTCAGA 85111 912819 1741 1756 25504 25519 AACCAACTCAGCTCAG 79 112 912820 17571772 25520 25535 TCCTAGCTTTTCATAA 23 113 912821 1788 1803 25551 25566TGCTGGACCGCTGCAC 0 114 912822 1796 1811 25559 25574 GAGTTAAGTGCTGGAC 93115 912823 1802 1817 25565 25580 GTATTAGAGTTAAGTG 92 116 912824 18031818 25566 25581 TGTATTAGAGTTAAGT 79 117 912825 1806 1821 25569 25584TGATGTATTAGAGTTA 92 118 912826 1808 1823 25571 25586 GCTGATGTATTAGAGT 80119 912827 1821 1836 25584 25599 TGAATTAACGCATGCT 83 120 912828 18221837 25585 25600 CTGAATTAACGCATGC 78 121 912829 1870 1885 25633 25648AGTAAGGGACCCTCTG 17 122 912830 1871 1886 25634 25649 CAGTAAGGGACCCTCT 28123 912831 1872 1887 25635 25650 TCAGTAAGGGACCCTC 77 124 912832 18741889 25637 25652 AGTCAGTAAGGGACCC 51 125 912833 1893 1908 25656 25671ATTAATAGGGCCACGA 80 126 912834 1895 1910 25658 25673 CCATTAATAGGGCCAC 90127 912835 1896 1911 25659 25674 ACCATTAATAGGGCCA 81 128 912836 19061921 25669 25684 GAACAGTCTGACCATT 82 129 912837 1908 1923 25671 25686TGGAACAGTCTGACCA 31 130 912838 1909 1924 25672 25687 CTGGAACAGTCTGACC 83131 912839 1911 1926 25674 25689 TGCTGGAACAGTCTGA 72 132 912840 19161931 25679 25694 CCTCATGCTGGAACAG 83 133 912841 1928 1943 25691 25706TCATTCTAAGAACCTC 96 134 912842 1945 1960 25708 25723 ACCCATCCAAACACCT 16135 912843 1982 1997 25745 25760 ACACATGGGCCAGCCT 70 136 912844 19892004 25752 25767 CAAGATCACACATGGG 70 137 912845 2057 2072 25820 25835GGGACGAACTGCACCC 0 138 912846 2098 2113 25861 25876 TATCATCTTTGCAGAC 81139 912847 2116 2131 25879 25894 GTTTTTAGTAGTCAAG 91 140 912848 21172132 25880 25895 CGTTTTTAGTAGTCAA 91 141 912849 2145 2160 25908 25923TATCATCTTGTTACCC 85 142 912850 2148 2163 25911 25926 GATTATCATCTTGTTA 70143 912851 2150 2165 25913 25928 TAGATTATCATCTTGT 53 144 912852 21512166 25914 25929 GTAGATTATCATCTTG 80 145 912853 2152 2167 25915 25930AGTAGATTATCATCTT 84 146 912854 2175 2190 25938 25953 GTGAAAAAGGTGTTCT 77147 912855 2182 2197 25945 25960 TAGTTAGGTGAAAAAG 92 148 912856 21882203 25951 25966 TTATTTTAGTTAGGTG 88 149 912857 2190 2205 25953 25968CATTATTTTAGTTAGG 86 150 912858 2273 2288 26036 26051 CTACTAACATCTCACT 55151 912859 2274 2289 26037 26052 TCTACTAACATCTCAC 89 152 912860 22782293 26041 26056 TTATTCTACTAACATC 27 153 912861 2280 2295 26043 26058GCTTATTCTACTAACA 79 154 912862 2281 2296 26044 26059 GGCTTATTCTACTAAC 81155 912863 2632 2647 26395 26410 GGTGAATGCCCTGCAC 41 156

TABLE 3 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 912864 2703 2718 26466 26481TTCAAGTTGTGTGCTC 90 157 912865 2755 2770 26518 26533 GGGAGAAACTCACTGA 37158 912866 N/A N/A 4416 4431 TGCTACTTGCCCCAGC 2 159 912867 N/A N/A 44214436 CACAATGCTACTTGCC 87 160 912868 N/A N/A 4584 4599 CCCAATGGCAGGGCTT58 161 912869 N/A N/A 4592 4607 TGCTCCTACCCAATGG 46 162 912870 N/A N/A4766 4781 GACTTTTATTGTTGCT 95 163 912871 N/A N/A 4883 4898TTCTATACCAGAGTGA 89 164 912872 N/A N/A 4884 4899 TTTCTATACCAGAGTG 89 165912873 N/A N/A 5405 5420 GTAGATGGCCTTAATG 83 166 912876 N/A N/A 61556170 TACATCCACGACTTCG 94 167 912877 N/A N/A 6156 6171 TTACATCCACGACTTC76 168 912880 N/A N/A 6606 6621 GGAACATTCAGGGTTT 13 169 912881 N/A N/A6834 6849 ATTACTTGGGTGCAGG 55 170 912884 N/A N/A 6838 6853GCAGATTACTTGGGTG 45 171 912885 N/A N/A 6931 6946 TGCAGGACAGGTTCCT 30 172912888 N/A N/A 7549 7564 CACACTGGGTCACCAC 55 173 912889 N/A N/A 75527567 AGTCACACTGGGTCAC 61 174 912928 N/A N/A 12273 12288 GGTATATGTTCCCAGG87 175 912929 N/A N/A 12314 12329 TATAACCACAGCCTGG 29 176 912932 N/A N/A12321 12336 CTGACTATATAACCAC 81 177 912933 N/A N/A 12666 12681ATCTTAGTGGCTGGGT 91 178 912936 N/A N/A 12767 12782 CTTACTATGGTAGAGT 88179 912937 N/A N/A 12768 12783 TCTTACTATGGTAGAG 74 180 912940 N/A N/A12835 12850 TGCATTGCATAGCCTT 97 181 912941 N/A N/A 12836 12851TTGCATTGCATAGCCT 96 182 912944 N/A N/A 12907 12922 TGCTTATAAAGCACAC 61183 912945 N/A N/A 12988 13003 GGAATAAGCCTCCACC 14 184 912948 N/A N/A14055 14070 GAAATCTGATTGCTTC 59 185 912949 N/A N/A 14393 14408TACTTATCTGCTCACT 66 186 912952 N/A N/A 14673 14688 TCTCTTAGTGTCCCCA 90187 14707 14722 912953 N/A N/A 14674 14689 ATCTCTTAGTGTCCCC 92 188 1470814723 912956 N/A N/A 15284 15299 TCACATTCATGCTTGC 82 189 912957 N/A N/A15291 15306 GATAACCTCACATTCA 0 190 912960 N/A N/A 15712 15727GAGCTAGGTGCTTCAC 6 191 912961 N/A N/A 15753 15768 ATAACAACTGAACCAC 85192 912964 N/A N/A 15937 15952 GTTATTAGCCAAATGC 92 193 912965 N/A N/A16468 16483 GGAGACTTGGCAAGGT 87 194 912968 N/A N/A 16960 16975ATTCATGACAGCCCTT 46 195 912969 N/A N/A 17128 17143 ATCGATTTTTCAGAGT 9196 912972 N/A N/A 17134 17149 ACAAACATCGATTTTT 52 197 912973 N/A N/A17769 17784 CTCTTTAATGACCTCG 90 198 912976 N/A N/A 18865 18880GTCAGAGGCACTCACA 25 199 912977 N/A N/A 18959 18974 AGCTATTATCTCCCAC 0200 912980 N/A N/A 19315 19330 AGTTTCTGGGCTTGCA 90 201 912981 N/A N/A19382 19397 GGCAATCACAAGAGAC 73 202 912984 N/A N/A 20286 20301AGAGGAAGCCCAATCA 79 203 20316 20331 912985 N/A N/A 20287 20302CAGAGGAAGCCCAATC 93 204 20317 20332 912988 N/A N/A 20658 20673TAGAAATTGCAGTGCC 92 205 912989 N/A N/A 20731 20746 TCCTATCCATATATTG 55206 912992 N/A N/A 21408 21423 GCAATTCTAGACATGG 88 207 912993 N/A N/A21558 21573 AGGACTTACACCAAGA 86 208 912996 N/A N/A 21936 21951TTCCTAATAAGAGCCC 24 209 912997 N/A N/A 21946 21961 GTCAAACATCTTCCTA 66210 913000 N/A N/A 22077 22092 AAAACTGTAGGATAGG 47 211 913001 N/A N/A22162 22177 GTTACATCCATAAAAC 0 212 913004 N/A N/A 22169 22184AGAGAATGTTACATCC 62 213 913008 N/A N/A 23083 23098 AAAGATTAATCAGGGC 61214 913012 N/A N/A 23788 23803 GTATTTACCTGGAGGC 0 215 913016 N/A N/A24426 24441 GGCCTATGATTTTCAG 0 216

TABLE 4 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 912874 N/A N/A 5869 5884ATACTTTTGGCAAGGC 96 217 912875 N/A N/A 5870 5885 AATACTTTTGGCAAGG 91 218912878 N/A N/A 6159 6174 TGCTTACATCCACGAC 12 219 912879 N/A N/A 62966311 CATCATGTTGGTCTCG 54 220 912882 N/A N/A 6835 6850 GATTACTTGGGTGCAG39 221 912883 N/A N/A 6837 6852 CAGATTACTTGGGTGC 69 222 912886 N/A N/A7083 7098 TTTAATGGTGTTTTGG 87 223 912887 N/A N/A 7478 7493TCAAATGCCGGTATTC 52 224 912890 N/A N/A 7587 7602 GTGAACTTCAACTTCC 56 225912930 N/A N/A 12317 12332 CTATATAACCACAGCC 77 226 912931 N/A N/A 1231912334 GACTATATAACCACAG 92 227 912934 N/A N/A 12670 12685AATCATCTTAGTGGCT 91 228 912935 N/A N/A 12765 12780 TACTATGGTAGAGTGG 80229 912938 N/A N/A 12786 12801 GTACATGGTCTGCAAA 84 230 912939 N/A N/A12787 12802 TGTACATGGTCTGCAA 57 231 912942 N/A N/A 12843 12858GCATGCATTGCATTGC 16 232 912943 N/A N/A 12885 12900 ACCAATCCTGTTAGAC 93233 912946 N/A N/A 13557 13572 GGAGACACCAAGCACC 42 234 912947 N/A N/A13751 13766 GCACTAAGTGTTAGAA 79 235 912950 N/A N/A 14396 14411GCTTACTTATCTGCTC 0 236 912951 N/A N/A 14501 14516 GGAGATCCATCCTGCA 0 237912954 N/A N/A 14675 14690 CATCTCTTAGTGTCCC 92 238 14709 14724 912955N/A N/A 15122 15137 TCCTAATGTCCTCAAC 9 239 912958 N/A N/A 15293 15308AAGATAACCTCACATT 33 240 912959 N/A N/A 15294 15309 CAAGATAACCTCACAT 22241 912962 N/A N/A 15754 15769 TATAACAACTGAACCA 82 242 912963 N/A N/A15856 15871 GCTTTAAAGCAGGACA 8 243 912966 N/A N/A 16774 16789AAAATTGTGGGTTTAG 68 244 912967 N/A N/A 16850 16865 ATCATTTGGACCATAG 81245 912970 N/A N/A 17130 17145 ACATCGATTTTTCAGA 83 246 912971 N/A N/A17133 17148 CAAACATCGATTTTTC 62 247 912974 N/A N/A 17843 17858GCTTTACAAGCTGGTC 0 248 912975 N/A N/A 17879 17894 ATCTATGTTCTCCTAG 0 249912978 N/A N/A 19125 19140 ACCTAAAATGCTCACC 0 250 912979 N/A N/A 1919819213 CCAGACTACATGCCAC 79 251 912982 N/A N/A 19446 19461TCTACTAGGCATCTCT 63 252 912983 N/A N/A 19447 19462 TTCTACTAGGCATCTC 42253 912986 N/A N/A 20288 20303 TCAGAGGAAGCCCAAT 92 254 20318 20333912987 N/A N/A 20656 20671 GAAATTGCAGTGCCCT 92 255 912990 N/A N/A 2139321408 GCCAACCTATCACTGA 60 256 912991 N/A N/A 21400 21415AGACATGGCCAACCTA 32 257 912994 N/A N/A 21565 21580 TGAAATAAGGACTTAC 67258 912995 N/A N/A 21934 21949 CCTAATAAGAGCCCCA 31 259 912998 N/A N/A22041 22056 GAAATCTGTCAGAGCA 33 260 912999 N/A N/A 22072 22087TGTAGGATAGGACTAG 0 261 913002 N/A N/A 22166 22181 GAATGTTACATCCATA 53262 913003 N/A N/A 22168 22183 GAGAATGTTACATCCA 80 263 913005 N/A N/A22605 22620 GTGATAAATCTGCAAG 70 264 913006 N/A N/A 23081 23096AGATTAATCAGGGCCA 8 265 913007 N/A N/A 23082 23097 AAGATTAATCAGGGCC 30266 913009 N/A N/A 23325 23340 GGTCACATGTGAGCCC 0 267 913010 N/A N/A23496 23511 CACTTCTGGTTCAAGA 13 268 913011 N/A N/A 23580 23595CCAATCTGATGACTTC 80 269 913013 N/A N/A 23790 23805 AAGTATTTACCTGGAG 0270 913014 N/A N/A 24028 24043 CACTCAAAGAGACTCA 65 271 913015 N/A N/A24425 24440 GCCTATGATTTTCAGG 0 272 913017 N/A N/A 24633 24648CACTACTGCCCTCTTC 50 273 913018 N/A N/A 24983 24998 TGCTGGGCTGATGTCA 0274 913019 N/A N/A 25150 25165 GGCATTTGGGACCTGA 67 275

TABLE 5 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 915343 1 16 2739 2754GCCCCCCTCGGACCAT 0 276 915363 45 60 2783 2798 CCTCAGTGTCTCGGCC 0 277915383 107 122 2845 2860 AATCGGCTCGGGTCCT 29 278 915403 190 205 29282943 GACAAGCTCCAGCCGC 64 279 915423 249 264 2987 3002 CGCTCAGGCAGCGGGT 0280 915443 347 362 N/A N/A CTCCAGCGGGATACCG 6 281 915463 386 401 59605975 GGCCTTCCGCACAAGA 0 282 915483 416 431 5990 6005 TGGATGGAAGATGCCA 28283 915503 452 467 6026 6041 GAGACCCTGTCGGAGG 45 284 915523 488 503 60626077 GATGAGCTGGTGGACA 70 285 915543 510 525 6084 6099 GAGAGATGCCTATTTT92 286 915563 559 574 6133 6148 GACCGAAAGTCAGACA 7 287 915603 697 71211915 11930 GTTTTGGCATCAATGA 94 288 915623 754 769 11972 11987GACTTGACTTTAGGGC 98 289 915643 827 842 12045 12060 CGAGAGAAGGTAGAGG 97290 915663 879 894 13615 13630 CTCGAAGGCATATCTC 66 291 915683 932 94716052 16067 GGGCCTGTTGCAGATG 0 292 915703 985 1000 16105 16120GGCATGGCGACCTCAG 6 293 915723 1037 1052 16157 16172 AGCCAAGGCAGCCGAC 0294 915743 1132 1147 16252 16267 GCGAGCCTGGGCGAGA 0 295 915763 1177 119219017 19032 CTCATGTATCCACCTT 88 296 915783 1229 1244 19069 19084GGGCAGCATTACATAA 73 297 915803 1286 1301 23691 23706 AAGCCATGTCACCAGT 34298 915823 1348 1363 23753 23768 ACTCGAGTGAACACCT 12 299 915843 14051420 25168 25183 GCCTGTTGGCTGCTCA 1 300 915863 1473 1488 25236 25251CTGCTGGACAGCCCTT 0 301 915883 1542 1557 25305 25320 CTTTATTGCCCAAGAA 72302 915903 1601 1616 25364 25379 CAGACTCTTCTCTAGT 49 303 915923 16331648 25396 25411 AATCTGCTAGACTCGC 88 304 915943 1686 1701 25449 25464CAGCAATGCGGAGGTA 80 305 915963 1768 1783 25531 25546 GAAAGGTTGCTTCCTA 84306 915983 1789 1804 25552 25567 GTGCTGGACCGCTGCA 11 307 916003 18151830 25578 25593 AACGCATGCTGATGTA 69 308 916023 1848 1863 25611 25626GCTTCCTGGTGTCATT 81 309 916043 1884 1899 25647 25662 GCCACGAAACAGTCAG 67310 916063 1913 1928 25676 25691 CATGCTGGAACAGTCT 20 311 916083 19541969 25717 25732 AAGGCCCCCACCCATC 0 312 916103 1977 1992 25740 25755TGGGCCAGCCTACCCC 0 313 916123 2026 2041 25789 25804 GGAAGTGGGATCATGC 55314 916142 2100 2115 25863 25878 GTTATCATCTTTGCAG 57 315 916162 21392154 25902 25917 CTTGTTACCCCCGCCA 84 316 916182 2264 2279 26027 26042TCTCACTGATTCACAT 83 317 916202 2624 2639 26387 26402 CCCTGCACACTAGATT 55318 916222 2677 2692 26440 26455 GAGGCGGAAGCTCCTG 0 319 916242 2707 272226470 26485 CAGGTTCAAGTTGTGT 83 320 916282 N/A N/A 4225 4240AAATGTACGGAATCTC 79 321 916302 N/A N/A 4822 4837 GTGTAAACATTTGTCC 74 322916322 N/A N/A 5414 5429 AGCTTTGGTGTAGATG 49 323 916342 N/A N/A 58015816 TACTATGGGAGCCACA 42 324 916362 N/A N/A 6866 6881 TGAAATTGTAACTGCC70 325 916382 N/A N/A 7492 7507 TAGATCGGTGCTGTTC 27 326 916402 N/A N/A7785 7800 GTTATAGGCGAGAGCA 0 327 916562 N/A N/A 12316 12331TATATAACCACAGCCT 58 328 916582 N/A N/A 12932 12947 ATAAGAGCTGTCTCCT 94329 916602 N/A N/A 13703 13718 CTAGTAAATGCTTGTC 96 330 916622 N/A N/A14177 14192 CTAATATTTCTACAGC 0 331 916642 N/A N/A 14672 14687CTCTTAGTGTCCCCAT 95 332 916662 N/A N/A 15542 15557 TTCCATCACAAGGCCT 50333 916682 N/A N/A 16317 16332 TCCATAATGCACAAGA 71 334 916702 N/A N/A17223 17238 TGTAGCTGGTTTGTGG 88 335 916722 N/A N/A 18223 18238AACAGCTACATCAGGC 44 336 916742 N/A N/A 19249 19264 GGCATTGCACATAGAC 74337 916761 N/A N/A 20410 20425 GTAAGCAATGCAGCCA 88 338 916781 N/A N/A20659 20674 TTAGAAATTGCAGTGC 91 339 916801 N/A N/A 20989 21004AGGTATTAAACTGCCA 25 340 916821 N/A N/A 21506 21521 GTCCTAAGAGCACTCA 57341 916841 N/A N/A 22603 22618 GATAAATCTGCAAGAG 49 342 916861 N/A N/A23472 23487 GGGACTTACACTGAAA 66 343 916881 N/A N/A 24314 24329GTCAACGCAGACTGCT 33 344

TABLE 6 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 915344 2 17 2740 2755CGCCCCCCTCGGACCA 0 345 915364 46 61 2784 2799 GCCTCAGTGTCTCGGC 0 346915384 108 123 2846 2861 GAATCGGCTCGGGTCC 49 347 915404 191 206 29292944 GGACAAGCTCCAGCCG 9 348 915424 250 265 2988 3003 TCGCTCAGGCAGCGGG 0349 915444 348 363 N/A N/A GCTCCAGCGGGATACC 0 350 915464 387 402 59615976 TGGCCTTCCGCACAAG 0 351 915484 428 443 6002 6017 TAAGTTGAAGGATGGA 96352 915504 453 468 6027 6042 AGAGACCCTGTCGGAG 80 353 915524 489 504 60636078 AGATGAGCTGGTGGAC 81 354 915544 512 527 6086 6101 AAGAGAGATGCCTATT77 355 915564 560 575 6134 6149 GGACCGAAAGTCAGAC 0 356 915604 700 71511918 11933 GTTGTTTTGGCATCAA 91 357 915624 755 770 11973 11988GGACTTGACTTTAGGG 81 358 915644 828 843 12046 12061 TCGAGAGAAGGTAGAG 24359 915664 880 895 13616 13631 CCTCGAAGGCATATCT 41 360 915684 952 96716072 16087 GATGACTTCAGGCCTG 0 361 915704 987 1002 16107 16122TGGGCATGGCGACCTC 0 362 915724 1038 1053 16158 16173 CAGCCAAGGCAGCCGA 0363 915744 1133 1148 16253 16268 AGCGAGCCTGGGCGAG 0 364 915764 1179 119419019 19034 TGCTCATGTATCCACC 56 365 915784 1230 1245 19070 19085AGGGCAGCATTACATA 69 366 915804 1293 1308 23698 23713 TATCTGGAAGCCATGT 6367 915824 1349 1364 23754 23769 CACTCGAGTGAACACC 0 368 915844 1406 142125169 25184 GGCCTGTTGGCTGCTC 0 369 915864 1477 1492 25240 25255GTCTCTGCTGGACAGC 0 370 915884 1545 1560 25308 25323 GTACTTTATTGCCCAA 73371 915904 1607 1622 25370 25385 GACTCACAGACTCTTC 92 372 915924 16341649 25397 25412 GAATCTGCTAGACTCG 65 373 915944 1687 1702 25450 25465ACAGCAATGCGGAGGT 83 374 915964 1769 1784 25532 25547 CGAAAGGTTGCTTCCT 79375 915984 1790 1805 25553 25568 AGTGCTGGACCGCTGC 38 376 916004 18161831 25579 25594 TAACGCATGCTGATGT 79 377 916024 1849 1864 25612 25627GGCTTCCTGGTGTCAT 73 378 916044 1885 1900 25648 25663 GGCCACGAAACAGTCA 40379 916064 1914 1929 25677 25692 TCATGCTGGAACAGTC 80 380 916084 19581973 25721 25736 TCACAAGGCCCCCACC 35 381 916104 1978 1993 25741 25756ATGGGCCAGCCTACCC 0 382 916124 2053 2068 25816 25831 CGAACTGCACCCCTTC 38383 916143 2101 2116 25864 25879 GGTTATCATCTTTGCA 81 384 916163 21402155 25903 25918 TCTTGTTACCCCCGCC 84 385 916183 2265 2280 26028 26043ATCTCACTGATTCACA 86 386 916203 2625 2640 26388 26403 GCCCTGCACACTAGAT 65387 916223 2678 2693 26441 26456 GGAGGCGGAAGCTCCT 0 388 916243 2709 272426472 26487 GCCAGGTTCAAGTTGT 62 389 916283 N/A N/A 4226 4241CAAATGTACGGAATCT 52 390 916303 N/A N/A 4864 4879 TACTTTAGGCTCCTGG 90 391916323 N/A N/A 5422 5437 AGCATTAGAGCTTTGG 75 392 916343 N/A N/A 58035818 TCTACTATGGGAGCCA 89 393 916363 N/A N/A 6927 6942 GGACAGGTTCCTTGGA 0394 916383 N/A N/A 7493 7508 CTAGATCGGTGCTGTT 14 395 916403 N/A N/A 77867801 AGTTATAGGCGAGAGC 0 396 916563 N/A N/A 12318 12333 ACTATATAACCACAGC90 397 916583 N/A N/A 12936 12951 GACAATAAGAGCTGTC 0 398 916603 N/A N/A13704 13719 GCTAGTAAATGCTTGT 73 399 916623 N/A N/A 14231 14246CCAACTTTTAGTATTA 92 400 916643 N/A N/A 14678 14693 AGCCATCTCTTAGTGT 50401 916663 N/A N/A 15566 15581 TCTGATGTCGAAGAGG 68 402 916683 N/A N/A16341 16356 TCCCATGTGGCAGTAC 0 403 916703 N/A N/A 17239 17254TCCAAATGCCCAACTC 37 404 916723 N/A N/A 18241 18256 GCAAATAATGTGCACA 22405 916743 N/A N/A 19250 19265 GGGCATTGCACATAGA 59 406 916762 N/A N/A20413 20428 GTAGTAAGCAATGCAG 69 407 916782 N/A N/A 20660 20675CTTAGAAATTGCAGTG 91 408 916802 N/A N/A 21002 21017 ATTTTAACAGCTCAGG 95409 916822 N/A N/A 21540 21555 TATGACATTTCAGAGT 88 410 916842 N/A N/A22629 22644 AGTACAAGCGCAGCCT 14 411 916862 N/A N/A 23538 23553ACAAGGACAAGCCCAC 37 412 916882 N/A N/A 24339 24354 GAAGTAGCGGCATCCC 68413

TABLE 7 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 915345 3 18 2741 2756CCGCCCCCCTCGGACC 0 414 915365 47 62 2785 2800 TGCCTCAGTGTCTCGG 0 415915385 109 124 2847 2862 GGAATCGGCTCGGGTC 72 416 915405 193 208 29312946 AAGGACAAGCTCCAGC 41 417 915425 251 266 2989 3004 CTCGCTCAGGCAGCGG 0418 915445 349 364 N/A N/A TGCTCCAGCGGGATAC 0 419 915465 388 403 59625977 CTGGCCTTCCGCACAA 16 420 915485 430 445 6004 6019 CTTAAGTTGAAGGATG27 421 915505 454 469 6028 6043 CAGAGACCCTGTCGGA 72 422 915525 492 5076066 6081 CGGAGATGAGCTGGTG 92 423 915545 513 528 6087 6102TAAGAGAGATGCCTAT 57 424 915565 561 576 6135 6150 TGGACCGAAAGTCAGA 0 425915605 701 716 11919 11934 GGTTGTTTTGGCATCA 97 426 915625 756 771 1197411989 TGGACTTGACTTTAGG 93 427 915645 829 844 12047 12062CTCGAGAGAAGGTAGA 0 428 915665 881 896 13617 13632 TCCTCGAAGGCATATC 0 429915685 953 968 16073 16088 GGATGACTTCAGGCCT 0 430 915705 988 1003 1610816123 CTGGGCATGGCGACCT 0 431 915725 1039 1054 16159 16174ACAGCCAAGGCAGCCG 0 432 915745 1134 1149 16254 16269 TAGCGAGCCTGGGCGA 0433 915765 1193 1208 19033 19048 CAAGTTGCAAATCTTG 0 434 915785 1231 124619071 19086 CAGGGCAGCATTACAT 74 435 915805 1300 1315 23705 23720TCGGGCATATCTGGAA 21 436 915825 1350 1365 23755 23770 GCACTCGAGTGAACAC 0437 915845 1407 1422 25170 25185 AGGCCTGTTGGCTGCT 0 438 915865 1480 149525243 25258 TTGGTCTCTGCTGGAC 21 439 915885 1546 1561 25309 25324GGTACTTTATTGCCCA 62 440 915905 1609 1624 25372 25387 GTGACTCACAGACTCT 81441 915925 1635 1650 25398 25413 AGAATCTGCTAGACTC 74 442 915945 16881703 25451 25466 CACAGCAATGCGGAGG 56 443 915965 1770 1785 25533 25548GCGAAAGGTTGCTTCC 66 444 915985 1791 1806 25554 25569 AAGTGCTGGACCGCTG 71445 916005 1817 1832 25580 25595 TTAACGCATGCTGATG 69 446 916025 18501865 25613 25628 GGGCTTCCTGGTGTCA 58 447 916045 1886 1901 25649 25664GGGCCACGAAACAGTC 9 448 916065 1915 1930 25678 25693 CTCATGCTGGAACAGT 86449 916085 1959 1974 25722 25737 ATCACAAGGCCCCCAC 82 450 916105 19791994 25742 25757 CATGGGCCAGCCTACC 0 451 916125 2054 2069 25817 25832ACGAACTGCACCCCTT 84 452 916144 2102 2117 25865 25880 AGGTTATCATCTTTGC 90453 916164 2141 2156 25904 25919 ATCTTGTTACCCCCGC 88 454 916184 22662281 26029 26044 CATCTCACTGATTCAC 91 455 916204 2626 2641 26389 26404TGCCCTGCACACTAGA 47 456 916224 2680 2695 26443 26458 GAGGAGGCGGAAGCTC 0457 916244 2710 2725 26473 26488 AGCCAGGTTCAAGTTG 71 458 916284 N/A N/A4227 4242 TCAAATGTACGGAATC 40 459 916304 N/A N/A 4865 4880GTACTTTAGGCTCCTG 89 460 916324 N/A N/A 5429 5444 A CATATCAGCATTAGA 87461 916344 N/A N/A 5804 5819 GTCTACTATGGGAGCC 90 462 916364 N/A N/A 69666981 GAAGATGCATAGAGGA 0 463 916384 N/A N/A 7550 7565 TCACACTGGGTCACCA 43464 916544 N/A N/A 12135 12150 GGCAATCAGGGAGGCA 32 465 916564 N/A N/A12320 12335 TGACTATATAACCACA 92 466 916584 N/A N/A 12951 12966CCCAATTGCCACTAGG 83 467 916604 N/A N/A 13718 13733 TCTTTACCAAGACCGC 92468 916624 N/A N/A 14245 14260 GACAAATTCATCAACC 87 469 916644 N/A N/A14778 14793 CTGTATCCAAAAGGCC 0 470 916664 N/A N/A 15597 15612ATACATAGCAGAGCCA 44 471 916684 N/A N/A 16352 16367 CACCCTATCGCTCCCA 43472 916704 N/A N/A 17267 17282 AGTTATGTCTGACTCA 72 473 916724 N/A N/A18254 18269 AATATACCCCACAGCA 40 474 916744 N/A N/A 19288 19303GTGCATGTGTGGCTTG 82 475 916763 N/A N/A 20414 20429 TGTAGTAAGCAATGCA 85476 916783 N/A N/A 20724 20739 CATATATTGCGGATGA 24 477 916803 N/A N/A21005 21020 GTTATTTTAACAGCTC 95 478 916823 N/A N/A 21561 21576ATAAGGACTTACACCA 83 479 916843 N/A N/A 22679 22694 CAGCATGCAACCACCC 8480 916863 N/A N/A 23550 23565 TGGGATGCTAGGACAA 72 481 916883 N/A N/A24340 24355 GGAAGTAGCGGCATCC 0 482

TABLE 8 Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targetingSEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2PNPLA3 SEQ Compound Start Stop Start Stop % ID Number Site Site SiteSite Sequence (5′ to 3′) Inhibition NO 915346 25 40 2763 2778CATTCCCAGCGCGACG 0 483 915366 52 67 2790 2805 TACCCTGCCTCAGTGT 0 484915386 112 127 2850 2865 TCGGGAATCGGCTCGG 26 485 915406 195 210 29332948 CGAAGGACAAGCTCCA 69 486 915426 252 267 2990 3005 GCTCGCTCAGGCAGCG 0487 915446 350 365 N/A N/A CTGCTCCAGCGGGATA 0 488 915466 389 404 59635978 CCTGGCCTTCCGCACA 40 489 915486 431 446 6005 6020 GCTTAAGTTGAAGGAT88 490 915506 455 470 6029 6044 GCAGAGACCCTGTCGG 32 491 915526 493 5086067 6082 CCGGAGATGAGCTGGT 4 492 915546 514 529 6088 6103GTAAGAGAGATGCCTA 94 493 915566 562 577 6136 6151 TTGGACCGAAAGTCAG 56 494915606 702 717 11920 11935 TGGTTGTTTTGGCATC 99 495 915626 757 772 1197511990 GTGGACTTGACTTTAG 89 496 915646 830 845 12048 12063TCTCGAGAGAAGGTAG 0 497 915666 882 897 13618 13633 ATCCTCGAAGGCATAT 0 498915686 956 971 16076 16091 TGAGGATGACTTCAGG 10 499 915706 989 1004 1610916124 GCTGGGCATGGCGACC 10 500 915726 1064 1079 16184 16199TAGCAGCTCATCTCCC 67 501 915746 1135 1150 16255 16270 GTAGCGAGCCTGGGCG 0502 915766 1196 1211 19036 19051 TAGCAAGTTGCAAATC 78 503 915786 12321247 19072 19087 ACAGGGCAGCATTACA 87 504 915806 1302 1317 23707 23722CGTCGGGCATATCTGG 53 505 915826 1352 1367 23757 23772 CAGCACTCGAGTGAAC 24506 915846 1408 1423 25171 25186 GAGGCCTGTTGGCTGC 0 507 915866 1508 152325271 25286 GAGGATGGACCGCGGG 0 508 915886 1549 1564 25312 25327GCAGGTACTTTATTGC 0 509 915906 1610 1625 25373 25388 AGTGACTCACAGACTC 35510 915926 1636 1651 25399 25414 AAGAATCTGCTAGACT 69 511 915946 16891704 25452 25467 ACACAGCAATGCGGAG 69 512 915966 1771 1786 25534 25549GGCGAAAGGTTGCTTC 58 513 915986 1792 1807 25555 25570 TAAGTGCTGGACCGCT 70514 916006 1818 1833 25581 25596 ATTAACGCATGCTGAT 73 515 916026 18511866 25614 25629 TGGGCTTCCTGGTGTC 71 516 916046 1887 1902 25650 25665AGGGCCACGAAACAGT 61 517 916066 1917 1932 25680 25695 ACCTCATGCTGGAACA 81518 916086 1960 1975 25723 25738 CATCACAAGGCCCCCA 48 519 916106 19801995 25743 25758 ACATGGGCCAGCCTAC 54 520 916126 2055 2070 25818 25833GACGAACTGCACCCCT 77 521 916145 2105 2120 25868 25883 TCAAGGTTATCATCTT 89522 916165 2142 2157 25905 25920 CATCTTGTTACCCCCG 89 523 916185 22702285 26033 26048 CTAACATCTCACTGAT 66 524 916205 2627 2642 26390 26405ATGCCCTGCACACTAG 62 525 916225 2681 2696 26444 26459 AGAGGAGGCGGAAGCT 25526 916245 2711 2726 26474 26489 AAGCCAGGTTCAAGTT 83 527 916285 N/A N/A4240 4255 ATTAGGACAAGATTCA 75 528 916305 N/A N/A 4866 4881TGTACTTTAGGCTCCT 93 529 916325 N/A N/A 5430 5445 AACATATCAGCATTAG 85 530916345 N/A N/A 5839 5854 CAAGGATGCCACCAAC 84 531 916365 N/A N/A 69746989 TCATTATGGAAGATGC 0 532 916385 N/A N/A 7602 7617 TTAACAACCCTGTCAG 1533 916545 N/A N/A 12151 12166 GTAACTGGTAGCTCCT 93 534 916565 N/A N/A12338 12353 ACCCATACTGCACCCC 79 535 916585 N/A N/A 12957 12972GCCTATCCCAATTGCC 70 536 916605 N/A N/A 13719 13734 GTCTTTACCAAGACCG 23537 916625 N/A N/A 14248 14263 AACGACAAATTCATCA 84 538 916645 N/A N/A14788 14803 TGCAATCCCCCTGTAT 17 539 916665 N/A N/A 15598 15613AATACATAGCAGAGCC 68 540 916685 N/A N/A 16366 16381 TGTCATGGTTGCCTCA 70541 916705 N/A N/A 17273 17288 ATAAGGAGTTATGTCT 80 542 916725 N/A N/A18255 18270 GAATATACCCCACAGC 58 543 916745 N/A N/A 19295 19310GTTACAGGTGCATGTG 75 544 916764 N/A N/A 20435 20450 AGTCATCTGGAGTCAC 69545 916784 N/A N/A 20756 20771 TCAGACAACCCACTGA 24 546 916804 N/A N/A21046 21061 AGGAATCTGAATCCTA 0 547 916824 N/A N/A 21640 21655GATAATTTCCTAGAGC 29 548 916844 N/A N/A 22699 22714 GAAATAAGTGCTCAGG 73549 916864 N/A N/A 23582 23597 CTCCAATCTGATGACT 53 550 916884 N/A N/A24347 24362 GAATTCAGGAAGTAGC 50 551

TABLE 9Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915347   26   41  2764  2779GCATTCCCAGCGCGAC  0 552 915367   58   73  2796  2811 GCTCTCTACCCTGCCT  9553 915387  113  128  2851  2866 ATCGGGAATCGGCTCG 28 554 915407  198 213  2936  2951 CCGCGAAGGACAAGCT  0 555 915427  253  268  2991  3006TGCTCGCTCAGGCAGC  0 556 915447  351  366 N/A N/A TCTGCTCCAGCGGGAT  1 557915467  391  406  5965  5980 CTCCTGGCCTTCCGCA 29 558 915487  433  448 6007  6022 TTGCTTAAGTTGAAGG 94 559 915507  456  471  6030  6045TGCAGAGACCCTGTCG 31 560 915527  494  509  6068  6083 GCCGGAGATGAGCTGG  0561 915547  515  530  6089  6104 GGTAAGAGAGATGCCT  0 562 915567  563 578  6137  6152 TTTGGACCGAAAGTCA  0 563 915607  703  718 11921 11936ATGGTTGTTTTGGCAT 35 564 915627  758  773 11976 11991 CGTGGACTTGACTTTA 85565 915647  831  846 12049 12064 CTCTCGAGAGAAGGTA  7 566 915667  883 898 13619 13634 TATCCTCGAAGGCATA  0 567 915687  959  974 16079 16094TTCTGAGGATGACTTC 39 568 915707  996 1011 16116 16131 TTGCCCAGCTGGGCAT  0569 915727 1065 1080 16185 16200 CTAGCAGCTCATCTCC 58 570 915747 11361151 16256 16271 TGTAGCGAGCCTGGGC 16 571 915767 1197 1212 19037 19052GTAGCAAGTTGCAAAT 80 572 915787 1233 1248 19073 19088 TACAGGGCAGCATTAC 71573 915807 1316 1331 23721 23736 CAACCACAGGACATCG  0 574 915827 13531368 23758 23773 TCAGCACTCGAGTGAA  0 575 915847 1409 1424 25172 25187GGAGGCCTGTTGGCTG  0 576 915867 1509 1524 25272 25287 TGAGGATGGACCGCGG 14577 915887 1553 1568 25316 25331 ACCAGCAGGTACTTTA 29 578 915907 16111626 25374 25389 AAGTGACTCACAGACT 29 579 915927 1637 1652 25400 25415AAAGAATCTGCTAGAC 60 580 915947 1690 1705 25453 25468 TACACAGCAATGCGGA 69581 915967 1772 1787 25535 25550 AGGCGAAAGGTTGCTT  0 582 915987 17931808 25556 25571 TTAAGTGCTGGACCGC 82 583 916007 1819 1834 25582 25597AATTAACGCATGCTGA 61 584 916027 1864 1879 25627 25642 GGACCCTCTGCACTGG 43585 916047 1888 1903 25651 25666 TAGGGCCACGAAACAG 80 586 916067 19181933 25681 25696 AACCTCATGCTGGAAC 72 587 916087 1961 1976 25724 25739CCATCACAAGGCCCCC 63 588 916107 1981 1996 25744 25759 CACATGGGCCAGCCTA 74589 916127 2056 2071 25819 25834 GGACGAACTGCACCCC  5 590 916146 21062121 25869 25884 GTCAAGGTTATCATCT 88 591 916166 2143 2158 25906 25921TCATCTTGTTACCCCC 90 592 916186 2272 2287 26035 26050 TACTAACATCTCACTG  1593 916206 2628 2643 26391 26406 AATGCCCTGCACACTA 56 594 916226 26822697 26445 26460 GAGAGGAGGCGGAAGC 10 595 916246 2712 2727 26475 26490TAAGCCAGGTTCAAGT 81 596 916286 N/A N/A  4244  4259 TTTCATTAGGACAAGA 61597 916306 N/A N/A  4867  4882 GTGTACTTTAGGCTCC 97 598 916326 N/A N/A 5431  5446 GAACATATCAGCATTA 52 599 916346 N/A N/A  5872  5887GTAATACTTTTGGCAA 75 600 916366 N/A N/A  7069  7084 GGTATTACAAATTATC 10601 916386 N/A N/A  7603  7618 CTTAACAACCCTGTCA  0 602 916546 N/A N/A12152 12167 AGTAACTGGTAGCTCC 88 603 916566 N/A N/A 12343 12358CTAATACCCATACTGC 84 604 916586 N/A N/A 12966 12981 AACTTTGCAGCCTATC 85605 916606 N/A N/A 13739 13754 AGAACTAAGGCAAATC 85 606 916626 N/A N/A14257 14272 GTCTTGGCCAACGACA  0 607 916646 N/A N/A 14793 14808CAGGATGCAATCCCCC 45 608 916666 N/A N/A 15601 15616 GCCAATACATAGCAGA 75609 916686 N/A N/A 16630 16645 GTCCATGAAATCCAGG  0 610 916706 N/A N/A17293 17308 TCTCTTAGGGCACCTC 87 611 916726 N/A N/A 18256 18271TGAATATACCCCACAG 24 612 916746 N/A N/A 19337 19352 AGCTCTAGGAGTCCCC 63613 916765 N/A N/A 20513 20528 CCAGATTGAGTCTCCT 91 614 916785 N/A N/A20775 20790 AATCAAGTGCCCTCCA 73 615 916805 N/A N/A 21211 21226TGTAGCTGTGTGGTGG 85 616 916825 N/A N/A 21760 21775 TACCATGATCAGGTCA  0617 916845 N/A N/A 22713 22728 GTAAAGATGTGAGTGA 85 618 916865 N/A N/A23606 23621 GTTTACAAAAGCTGCC 17 619 916885 N/A N/A 24375 24390TGAACTCCGGCTCAGT  0 620

TABLE 10Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915348   28   43  2766  2781GGGCATTCCCAGCGCG  0 621 915368   59   74  2797  2812 CGCTCTCTACCCTGCC  0622 915388  114  129  2852  2867 GATCGGGAATCGGCTC 32 623 915408  199 214  2937  2952 CCCGCGAAGGACAAGC  6 624 915428  275  290  3013  3028GTCGCGGAGGAGGTGC  0 625 915448  352  367 N/A N/A GTCTGCTCCAGCGGGA  4 626915468  392  407  5966  5981 ACTCCTGGCCTTCCGC 87 627 915488  434  449 6008  6023 CTTGCTTAAGTTGAAG  0 628 915508  457  472  6031  6046TTGCAGAGACCCTGTC 63 629 915528  495  510  6069  6084 TGCCGGAGATGAGCTG  0630 915548  516  531  6090  6105 TGGTAAGAGAGATGCC 18 631 915568  564 579  6138  6153 CTTTGGACCGAAAGTC 10 632 915608  704  719 11922 11937GATGGTTGTTTTGGCA 98 633 915628  772  787 11990 12005 ACATGAAGAAAGTTCG 41634 915648  832  847 12050 12065 GCTCTCGAGAGAAGGT 55 635 915668  884 899 13620 13635 ATATCCTCGAAGGCAT 33 636 915688  962  977 16082 16097CCCTTCTGAGGATGAC 11 637 915708  998 1013 16118 16133 GTTTGCCCAGCTGGGC  0638 915728 1067 1082 16187 16202 GTCTAGCAGCTCATCT 68 639 915748 11371152 16257 16272 CTGTAGCGAGCCTGGG  0 640 915768 1198 1213 19038 19053GGTAGCAAGTTGCAAA 90 641 915788 1234 1249 19074 19089 GTACAGGGCAGCATTA 69642 915808 1317 1332 23722 23737 GCAACCACAGGACATC 51 643 915828 13541369 23759 23774 ATCAGCACTCGAGTGA  0 644 915848 1410 1425 25173 25188GGGAGGCCTGTTGGCT 17 645 915868 1510 1525 25273 25288 CTGAGGATGGACCGCG 53646 915888 1554 1569 25317 25332 CACCAGCAGGTACTTT  0 647 915908 16121627 25375 25390 CAAGTGACTCACAGAC 91 648 915928 1639 1654 25402 25417TGAAAGAATCTGCTAG 59 649 915948 1691 1706 25454 25469 CTACACAGCAATGCGG 20650 915968 1773 1788 25536 25551 CAGGCGAAAGGTTGCT 60 651 915988 17941809 25557 25572 GTTAAGTGCTGGACCG 86 652 916008 1820 1835 25583 25598GAATTAACGCATGCTG 88 653 916028 1865 1880 25628 25643 GGGACCCTCTGCACTG  0654 916048 1889 1904 25652 25667 ATAGGGCCACGAAACA 75 655 916068 19191934 25682 25697 GAACCTCATGCTGGAA 72 656 916088 1962 1977 25725 25740CCCATCACAAGGCCCC 37 657 916108 1984 1999 25747 25762 TCACACATGGGCCAGC 84658 916128 2079 2094 25842 25857 CTGACAGGCAGTGTCG 10 659 916147 21072122 25870 25885 AGTCAAGGTTATCATC 81 660 916167 2144 2159 25907 25922ATCATCTTGTTACCCC 88 661 916187 2276 2291 26039 26054 ATTCTACTAACATCTC 90662 916207 2629 2644 26392 26407 GAATGCCCTGCACACT 72 663 916227 26912706 26454 26469 GCTCCAGTGGAGAGGA 14 664 916247 2713 2728 26476 26491ATAAGCCAGGTTCAAG 88 665 916287 N/A N/A  4308  4323 GTGAGAAACAAACCCT 92666 916307 N/A N/A  4882  4897 TCTATACCAGAGTGAG 84 667 916327 N/A N/A 5514  5529 AGGAATGAGTCTCCCA 17 668 916347 N/A N/A  5873  5888GGTAATACTTTTGGCA 70 669 916367 N/A N/A  7106  7121 CGCTTATGAAAGCATC  0670 916387 N/A N/A  7605  7620 CCCTTAACAACCCTGT 28 671 916547 N/A N/A12167 12182 TTTGATTGTGCAGACA 98 672 916567 N/A N/A 12345 12360TCCTAATACCCATACT 74 673 916587 N/A N/A 12969 12984 ACAAACTTTGCAGCCT 95674 916607 N/A N/A 13742 13757 GTTAGAACTAAGGCAA 94 675 916627 N/A N/A14301 14316 GAGCAGATAAATACAC 91 676 916647 N/A N/A 14892 14907TGGTATCTCGCTTCCT  0 677 916667 N/A N/A 15613 15628 TAAAGCCACGCAGCCA 46678 916687 N/A N/A 16656 16671 CCAGATGCAGGACCCC  0 679 916707 N/A N/A17326 17341 AAACTAATGCACCTGG 43 680 916727 N/A N/A 18257 18272CTGAATATACCCCACA 75 681 916747 N/A N/A 19360 19375 AGCTGCTATGTGAGGC 12682 916766 N/A N/A 20520 20535 TCAGTAACCAGATTGA 25 683 916786 N/A N/A20778 20793 TTTAATCAAGTGCCCT 81 684 916806 N/A N/A 21216 21231CAGGATGTAGCTGTGT 84 685 916826 N/A N/A 21887 21902 TAAGATCCCATCTTAC 13686 916846 N/A N/A 22739 22754 AAAGTAAACACCCACC 42 687 916866 N/A N/A23625 23640 GCTTACAACACTACCC 57 688 916886 N/A N/A 24393 24408GTAATGGGAGCCAGGC 38 689

TABLE 11Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915349   29   44  2767  2782AGGGCATTCCCAGCGC  0 690 915369   60   75  2798  2813 GCGCTCTCTACCCTGC 23691 915389  115  130  2853  2868 GGATCGGGAATCGGCT 54 692 915409  200 215  2938  2953 GCCCGCGAAGGACAAG 32 693 915429  276  291  3014  3029CGTCGCGGAGGAGGTG  0 694 915449  364  379  5938  5953 AGGACCTGCAGAGTCT 21695 915469  394  409  5968  5983 CGACTCCTGGCCTTCC 59 696 915489  435 450  6009  6024 ACTTGCTTAAGTTGAA 86 697 915509  466  481  6040  6055GGGAGGCATTTGCAGA 57 698 915529  496  511  6070  6085 TTGCCGGAGATGAGCT 40699 915549  518  533  6092  6107 TCTGGTAAGAGAGATG 61 700 915569  565 580  6139  6154 TCTTTGGACCGAAAGT  9 701 915609  705  720 11923 11938TGATGGTTGTTTTGGC 99 702 915629  776  791 11994 12009 GTCCACATGAAGAAAG 32703 915649  833  848 12051 12066 AGCTCTCGAGAGAAGG 36 704 915669  885 900 13621 13636 AATATCCTCGAAGGCA 55 705 915689  969  984 16089 16104GATCCATCCCTTCTGA 20 706 915709  999 1014 16119 16134 TGTTTGCCCAGCTGGG  5707 915729 1077 1092 16197 16212 GACGCAGGTGGTCTAG  0 708 915749 11381153 N/A N/A GCTGTAGCGAGCCTGG 71 709 915769 1200 1215 19040 19055TGGGTAGCAAGTTGCA 81 710 915789 1235 1250 19075 19090 GGTACAGGGCAGCATT 88711 915809 1318 1333 23723 23738 TGCAACCACAGGACAT 40 712 915829 13551370 23760 23775 CATCAGCACTCGAGTG  0 713 915849 1424 1439 25187 25202CTCAGGTGTGCATGGG 61 714 915869 1511 1526 25274 25289 CCTGAGGATGGACCGC 70715 915889 1556 1571 25319 25334 AGCACCAGCAGGTACT 35 716 915909 16131628 25376 25391 TCAAGTGACTCACAGA 84 717 915929 1645 1660 25408 25423CACCTCTGAAAGAATC 89 718 915949 1692 1707 25455 25470 ACTACACAGCAATGCG 33719 915969 1774 1789 25537 25552 ACAGGCGAAAGGTTGC 88 720 915989 17951810 25558 25573 AGTTAAGTGCTGGACC 84 721 916009 1823 1838 25586 25601GCTGAATTAACGCATG 67 722 916029 1866 1881 25629 25644 AGGGACCCTCTGCACT 15723 916049 1890 1905 25653 25668 AATAGGGCCACGAAAC 52 724 916069 19201935 25683 25698 AGAACCTCATGCTGGA 85 725 916089 1963 1978 25726 25741CCCCATCACAAGGCCC 20 726 916109 1985 2000 25748 25763 ATCACACATGGGCCAG 72727 916129 2080 2095 25843 25858 CCTGACAGGCAGTGTC 15 728 916148 21082123 25871 25886 TAGTCAAGGTTATCAT 87 729 916168 2146 2161 25909 25924TTATCATCTTGTTACC 82 730 916188 2279 2294 26042 26057 CTTATTCTACTAACAT 87731 916208 2630 2645 26393 26408 TGAATGCCCTGCACAC 68 732 916228 26922707 26455 26470 TGCTCCAGTGGAGAGG 80 733 916248 2726 2741 26489 26504GTCCCTGCAGAAAATA  0 734 916288 N/A N/A  4337  4352 AGCATACCACACCCCA 75735 916308 N/A N/A  5086  5101 GGACATGCTCAGCAGC 68 736 916328 N/A N/A 5533  5548 TGCTGTAGGCCTCAGC  0 737 916348 N/A N/A  5874  5889TGGTAATACTTTTGGC 86 738 916368 N/A N/A  7132  7147 GTAAATGGAGTCCTTC 80739 916388 N/A N/A  7612  7627 CATAATCCCCTTAACA 32 740 916548 N/A N/A12195 12210 TTAACCATCAAGGACA 77 741 916568 N/A N/A 12665 12680TCTTAGTGGCTGGGTA 85 742 916588 N/A N/A 12973 12988 CCTAACAAACTTTGCA 32743 916608 N/A N/A 13749 13764 ACTAAGTGTTAGAACT 76 744 916628 N/A N/A14338 14353 CTGCAGTATCCCTAGC  0 745 916648 N/A N/A 15012 15027TCCCATCGGTCATTTC 45 746 916668 N/A N/A 15682 15697 GAAACCACTATCATCA 62747 916688 N/A N/A 16671 16686 GTAATAGGCCAAGTCC  0 748 916708 N/A N/A17327 17342 CAAACTAATGCACCTG 66 749 916728 N/A N/A 18332 18347CCAATATCATAGCTGA 85 750 916748 N/A N/A 19376 19391 CACAAGAGACTGGACC 64751 916767 N/A N/A 20551 20566 TACTATGGGATGAGTA  0 752 916787 N/A N/A20779 20794 TTTTAATCAAGTGCCC 38 753 916807 N/A N/A 21218 21233GGCAGGATGTAGCTGT 63 754 916827 N/A N/A 21947 21962 AGTCAAACATCTTCCT 50755 916847 N/A N/A 22759 22774 CAGACTAACTTACTAA 77 756 916867 N/A N/A23626 23641 AGCTTACAACACTACC 13 757 916887 N/A N/A 24505 24520ATGCTACGGGCTCTCA  0 758

TABLE 12Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915350   30   45  2768  2783CAGGGCATTCCCAGCG  0 759 915370   82   97  2820  2835 CAGCTCCGCCCGGCGC 14760 915390  130  145  2868  2883 TTAGGATCTGGGTCGG 88 761 915410  201 216  2939  2954 AGCCCGCGAAGGACAA  0 762 915430  295  310  3033  3048GCGCCGAACAACATGC  0 763 915450  366  381  5940  5955 AGAGGACCTGCAGAGT 83764 915470  395  410  5969  5984 CCGACTCCTGGCCTTC 68 765 915490  436 451  6010  6025 AACTTGCTTAAGTTGA 41 766 915510  467  482  6041  6056CGGGAGGCATTTGCAG 44 767 915530  497  512  6071  6086 TTTGCCGGAGATGAGC 92768 915550  519  534  6093  6108 CTCTGGTAAGAGAGAT 20 769 915570  566 581  6140  6155 GTCTTTGGACCGAAAG 19 770 915590  627  642  7857  7872GAGGGATAAGGCCACT 83 771 915610  706  721 11924 11939 GTGATGGTTGTTTTGG 97772 915630  782  797 12000 12015 GGTGATGTCCACATGA 87 773 915650  834 849 12052 12067 AAGCTCTCGAGAGAAG 44 774 915670  887  902 13623 13638CAAATATCCTCGAAGG  0 775 915690  970  985 16090 16105 GGATCCATCCCTTCTG  0776 915710 1003 1018 16123 16138 CTCATGTTTGCCCAGC 68 777 915730 10781093 16198 16213 AGACGCAGGTGGTCTA  0 778 915750 1139 1154 N/A N/ATGCTGTAGCGAGCCTG 56 779 915770 1201 1216 19041 19056 ATGGGTAGCAAGTTGC 79780 915790 1247 1262 19087 19102 TTCCACAGGCAGGGTA 48 781 915810 13201335 23725 23740 ACTGCAACCACAGGAC 22 782 915830 1356 1371 23761 23776ACATCAGCACTCGAGT  0 783 915850 1427 1442 25190 25205 CTGCTCAGGTGTGCAT 10784 915870 1512 1527 25275 25290 ACCTGAGGATGGACCG 69 785 915890 15571572 25320 25335 CAGCACCAGCAGGTAC 62 786 915910 1617 1632 25380 25395CTCCTCAAGTGACTCA 83 787 915930 1648 1663 25411 25426 TAGCACCTCTGAAAGA 55788 915950 1693 1708 25456 25471 CACTACACAGCAATGC 74 789 915970 17751790 25538 25553 CACAGGCGAAAGGTTG 72 790 915990 1797 1812 25560 25575AGAGTTAAGTGCTGGA 92 791 916010 1824 1839 25587 25602 AGCTGAATTAACGCAT  0792 916030 1867 1882 25630 25645 AAGGGACCCTCTGCAC 38 793 916050 18911906 25654 25669 TAATAGGGCCACGAAA 53 794 916070 1921 1936 25684 25699AAGAACCTCATGCTGG 24 795 916090 1964 1979 25727 25742 CCCCCATCACAAGGCC 24796 916110 1986 2001 25749 25764 GATCACACATGGGCCA  0 797 916130 20812096 25844 25859 ACCTGACAGGCAGTGT 54 798 916149 2109 2124 25872 25887GTAGTCAAGGTTATCA 87 799 916169 2154 2169 25917 25932 TAAGTAGATTATCATC 79800 916189 2282 2297 26045 26060 AGGCTTATTCTACTAA 85 801 916209 26312646 26394 26409 GTGAATGCCCTGCACA 59 802 916229 2693 2708 26456 26471GTGCTCCAGTGGAGAG 54 803 916249 2727 2742 26490 26505 GGTCCCTGCAGAAAAT 38804 916289 N/A N/A  4338  4353 AAGCATACCACACCCC 79 805 916309 N/A N/A 5278  5293 AATCTTGGGATGCACA 95 806 916329 N/A N/A  5569  5584CATCATGGCTTCCAGT 79 807 916349 N/A N/A  5879  5894 TGGGATGGTAATACTT  0808 916369 N/A N/A  7134  7149 AAGTAAATGGAGTCCT  5 809 916389 N/A N/A 7615  7630 TTGCATAATCCCCTTA 33 810 916409 N/A N/A  8165  8180TTAACTAGATCACTGA 58 811 916429 N/A N/A  9109  9124 TCCTAATGCGAGTCCC 86812 916449 N/A N/A  9522  9537 TGCTGCTGGGTGCACT 45 813 916469 N/A N/A10199 10214 GGTGATGACACAGCAT 94 814 916489 N/A N/A 10382 10397GCCATGTACAACTTTT 52 815 916509 N/A N/A 11152 11167 TACAATTTGGACAGAG 71816 916529 N/A N/A 11546 11561 ACCTATAGGAGTGCCC 35 817 916549 N/A N/A12204 12219 TTATTTCCGTTAACCA 97 818 916569 N/A N/A 12672 12687AGAATCATCTTAGTGG 94 819 916589 N/A N/A 12989 13004 CGGAATAAGCCTCCAC  0820 916609 N/A N/A 13752 13767 GGCACTAAGTGTTAGA 57 821 916629 N/A N/A14375 14390 TCTCACAAGGCTGGCA 84 822 916649 N/A N/A 15137 15152GCCATACCGGCTCCCT 30 823 916669 N/A N/A 15691 15706 GGCCTTACAGAAACCA 15824 916689 N/A N/A 16672 16687 AGTAATAGGCCAAGTC 16 825 916709 N/A N/A17328 17343 ACAAACTAATGCACCT 42 826 916729 N/A N/A 18333 18348TCCAATATCATAGCTG 32 827 916749 N/A N/A 19445 19460 CTACTAGGCATCTCTA 32828 916768 N/A N/A 20553 20568 CTTACTATGGGATGAG 83 829 916788 N/A N/A20808 20823 TAATATTCAGACCAGG 94 830 916808 N/A N/A 21252 21267CCATGCATGGCACAGT  4 831 916828 N/A N/A 21968 21983 AGACAGGAATCCAACC  0832 916848 N/A N/A 22767 22782 GGACATGACAGACTAA 96 833 916868 N/A N/A23637 23652 GCAGACACAACAGCTT 40 834 916888 N/A N/A 24507 24522CCATGCTACGGGCTCT  0 835

TABLE 13Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915351   33   48  2771  2786GGCCAGGGCATTCCCA  0 836 915371   83   98  2821  2836 GCAGCTCCGCCCGGCG  2837 915391  132  147  2870  2885 GGTTAGGATCTGGGTC 54 838 915411  222 237  2960  2975 GGTAGAAGCCCAGGAA 57 839 915431  321  336  3059  3074CGACGCAGTGCAACGC 49 840 915451  368  383  5942  5957 TGAGAGGACCTGCAGA  0841 915471  400  415  5974  5989 ATGTTCCGACTCCTGG 82 842 915491  437 452  6011  6026 GAACTTGCTTAAGTTG 23 843 915511  468  483  6042  6057CCGGGAGGCATTTGCA  0 844 915531  498  513  6072  6087 TTTTGCCGGAGATGAG 84845 915551  520  535  6094  6109 ACTCTGGTAAGAGAGA  5 846 915571  567 582  6141  6156 CGTCTTTGGACCGAAA 64 847 915611  708  723 11926 11941CGGTGATGGTTGTTTT 98 848 915631  783  798 12001 12016 TGGTGATGTCCACATG  0849 915651  835  850 12053 12068 AAAGCTCTCGAGAGAA 35 850 915671  890 905 13626 13641 ATCCAAATATCCTCGA 42 851 915691  971  986 16091 16106AGGATCCATCCCTTCT  0 852 915711 1005 1020 16125 16140 GACTCATGTTTGCCCA 73853 915731 1079 1094 16199 16214 GAGACGCAGGTGGTCT  0 854 915751 11401155 N/A N/A GTGCTGTAGCGAGCCT  0 855 915771 1202 1217 19042 19057AATGGGTAGCAAGTTG 80 856 915791 1248 1263 19088 19103 ATTCCACAGGCAGGGT 37857 915811 1327 1342 23732 23747 GTCACCCACTGCAACC 52 858 915831 13571372 23762 23777 CACATCAGCACTCGAG 29 859 915851 1429 1444 25192 25207TCCTGCTCAGGTGTGC  2 860 915871 1513 1528 25276 25291 GACCTGAGGATGGACC 20861 915891 1558 1573 25321 25336 TCAGCACCAGCAGGTA 68 862 915911 16201635 25383 25398 CGCCTCCTCAAGTGAC 48 863 915931 1652 1667 25415 25430ACTTTAGCACCTCTGA 93 864 915951 1695 1710 25458 25473 GTCACTACACAGCAAT 84865 915971 1776 1791 25539 25554 GCACAGGCGAAAGGTT 74 866 915991 17981813 25561 25576 TAGAGTTAAGTGCTGG 84 867 916011 1825 1840 25588 25603CAGCTGAATTAACGCA  0 868 916031 1868 1883 25631 25646 TAAGGGACCCTCTGCA 54869 916051 1892 1907 25655 25670 TTAATAGGGCCACGAA 75 870 916071 19221937 25685 25700 TAAGAACCTCATGCTG 56 871 916091 1965 1980 25728 25743CCCCCCATCACAAGGC  9 872 916111 1987 2002 25750 25765 AGATCACACATGGGCC 26873 916131 2084 2099 25847 25862 ACCACCTGACAGGCAG 80 874 916150 21102125 25873 25888 AGTAGTCAAGGTTATC 92 875 916170 2174 2189 25937 25952TGAAAAAGGTGTTCTA 49 876 916190 2283 2298 26046 26061 AAGGCTTATTCTACTA 79877 916210 2633 2648 26396 26411 AGGTGAATGCCCTGCA 71 878 916230 26942709 26457 26472 TGTGCTCCAGTGGAGA 75 879 916250 2728 2743 26491 26506TGGTCCCTGCAGAAAA 79 880 916290 N/A N/A  4397  4412 TGCCTACTGGCTCACA 14881 916310 N/A N/A  5279  5294 AAATCTTGGGATGCAC 94 882 916330 N/A N/A 5572  5587 TGACATCATGGCTTCC 93 883 916350 N/A N/A  6158  6173GCTTACATCCACGACT  0 884 916370 N/A N/A  7135  7150 CAAGTAAATGGAGTCC 77885 916390 N/A N/A  7620  7635 ATCTATTGCATAATCC 86 886 916550 N/A N/A12205 12220 TTTATTTCCGTTAACC 96 887 916570 N/A N/A 12694 12709TTCTTGACCGTGTTTC 98 888 916590 N/A N/A 12990 13005 CCGGAATAAGCCTCCA 47889 916610 N/A N/A 13822 13837 TGTACAATGGGACGGA 69 890 916630 N/A N/A14418 14433 ATCGACACAGCATCAC 92 891 916650 N/A N/A 15138 15153TGCCATACCGGCTCCC  0 892 916670 N/A N/A 15758 15773 GGTTTATAACAACTGA 89893 916690 N/A N/A 16722 16737 GCCTTGAGGTGGGTGG  0 894 916710 N/A N/A17512 17527 AGTCATGGGATGTGCA 58 895 916730 N/A N/A 18395 18410ATGTTTGGAAGTCGCC 92 896 916750 N/A N/A 19473 19488 AAGGATCCTGCTTCTA  9897 916769 N/A N/A 20554 20569 GCTTACTATGGGATGA 62 898 916789 N/A N/A20809 20824 GTAATATTCAGACCAG 96 899 916809 N/A N/A 21254 21269ATCCATGCATGGCACA 72 900 916829 N/A N/A 21979 21994 GTCAGACACGGAGACA  0901 916849 N/A N/A 23110 23125 GGCTTTTGAAGGAGAG 84 902 916869 N/A N/A23787 23802 TATTTACCTGGAGGCG  0 903 916889 N/A N/A 24612 24627CAAATCGGATCTTTGC 44 904

TABLE 14Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915352   34   49  2772  2787CGGCCAGGGCATTCCC  0 905 915372   86  101  2824  2839 GCAGCAGCTCCGCCCG 48906 915392  135  150  2873  2888 GCGGGTTAGGATCTGG 25 907 915412  225 240  2963  2978 CGTGGTAGAAGCCCAG  2 908 915432  322  337  3060  3075CCGACGCAGTGCAACG 31 909 915452  371  386  5945  5960 ATCTGAGAGGACCTGC 72910 915472  401  416  5975  5990 AATGTTCCGACTCCTG 77 911 915492  438 453  6012  6027 GGAACTTGCTTAAGTT 55 912 915512  469  484  6043  6058GCCGGGAGGCATTTGC  3 913 915532  499  514  6073  6088 ATTTTGCCGGAGATGA 86914 915552  521  536  6095  6110 CACTCTGGTAAGAGAG  8 915 915612  709 724 11927 11942 ACGGTGATGGTTGTTT 87 916 915632  787  802 12005 12020AGCTTGGTGATGTCCA 38 917 915652  836  851 12054 12069 AAAAGCTCTCGAGAGA  0918 915672  891  906 13627 13642 CATCCAAATATCCTCG 82 919 915692  972 987 16092 16107 CAGGATCCATCCCTTC  0 920 915712 1006 1021 16126 16141AGACTCATGTTTGCCC 77 921 915732 1081 1096 16201 16216 CTGAGACGCAGGTGGT 87922 915752 1141 1156 N/A N/A AGTGCTGTAGCGAGCC  1 923 915772 1203 121819043 19058 TAATGGGTAGCAAGTT 77 924 915792 1257 1272 19097 19112CAATGGCAGATTCCAC 56 925 915812 1328 1343 23733 23748 GGTCACCCACTGCAAC 58926 915832 1358 1373 23763 23778 ACACATCAGCACTCGA 66 927 915852 14301445 25193 25208 GTCCTGCTCAGGTGTG 52 928 915872 1518 1533 25281 25296GGCTGGACCTGAGGAT  0 929 915892 1570 1585 25333 25348 GTGGAGAGCCCCTCAG 47930 915912 1621 1636 25384 25399 TCGCCTCCTCAAGTGA  8 931 915932 16541669 25417 25432 AAACTTTAGCACCTCT 90 932 915952 1696 1711 25459 25474GGTCACTACACAGCAA 82 933 915972 1777 1792 25540 25555 TGCACAGGCGAAAGGT 64934 915992 1799 1814 25562 25577 TTAGAGTTAAGTGCTG 91 935 916012 18261841 25589 25604 CCAGCTGAATTAACGC 32 936 916032 1869 1884 25632 25647GTAAGGGACCCTCTGC 73 937 916052 1894 1909 25657 25672 CATTAATAGGGCCACG 81938 916072 1923 1938 25686 25701 CTAAGAACCTCATGCT 70 939 916092 19661981 25729 25744 ACCCCCCATCACAAGG 30 940 916112 1988 2003 25751 25766AAGATCACACATGGGC 86 941 916132 2085 2100 25848 25863 GACCACCTGACAGGCA 61942 916151 2111 2126 25874 25889 TAGTAGTCAAGGTTAT 88 943 916171 21762191 25939 25954 GGTGAAAAAGGTGTTC 84 944 916191 2284 2299 26047 26062TAAGGCTTATTCTACT 76 945 916211 2634 2649 26397 26412 GAGGTGAATGCCCTGC  0946 916231 2695 2710 26458 26473 GTGTGCTCCAGTGGAG 87 947 916251 27292744 26492 26507 CTGGTCCCTGCAGAAA 67 948 916291 N/A N/A  4419  4434CAATGCTACTTGCCCC 68 949 916311 N/A N/A  5280  5295 TAAATCTTGGGATGCA 94950 916331 N/A N/A  5576  5591 ACAATGACATCATGGC 97 951 916351 N/A N/A 6165  6180 GCAAACTGCTTACATC  0 952 916371 N/A N/A  7172  7187GTTAGACGCGCCAGGC  7 953 916391 N/A N/A  7624  7639 TCTCATCTATTGCATA  0954 916551 N/A N/A 12206 12221 TTTTATTTCCGTTAAC 73 955 916571 N/A N/A12714 12729 TAAACTACCGAACGCA 96 956 916591 N/A N/A 12991 13006CCCGGAATAAGCCTCC 47 957 916611 N/A N/A 13823 13838 CTGTACAATGGGACGG 23958 916631 N/A N/A 14422 14437 TCCCATCGACACAGCA 95 959 916651 N/A N/A15206 15221 GGAATATTGCCAGGTA 95 960 916671 N/A N/A 15759 15774TGGTTTATAACAACTG 29 961 916691 N/A N/A 16746 16761 ATTAGGAGAGGTCTCA 55962 916711 N/A N/A 17602 17617 CTTGATAGTGAATGTG 90 963 916731 N/A N/A18859 18874 GGCACTCACAAAAGCG 10 964 916751 N/A N/A 20182 20197CCCTATGTTCTACTTT 54 965 916770 N/A N/A 20572 20587 CAACATCTCTAGCTGG 82966 916790 N/A N/A 20810 20825 GGTAATATTCAGACCA  0 967 916810 N/A N/A21265 21280 TGAAGCTACAGATCCA 74 968 916830 N/A N/A 22042 22057GGAAATCTGTCAGAGC 18 969 916850 N/A N/A 23142 23157 GAATCTAGGAAGGCGA 77970 916870 N/A N/A 23789 23804 AGTATTTACCTGGAGG  0 971 916890 N/A N/A24738 24753 AGCCTTAGGAAGCCTC 16 972

TABLE 15Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915353   35   50  2773  2788TCGGCCAGGGCATTCC  0  973 915373   87  102  2825  2840 CGCAGCAGCTCCGCCC 0  974 915393  136  151  2874  2889 CGCGGGTTAGGATCTG  0  975 915413 239  254  2977  2992 GCGGGTCGCCCCGACG  0  976 915433  325  340  3063 3078 ACGCCGACGCAGTGCA  0  977 915453  372  387  5946  5961GATCTGAGAGGACCTG 24  978 915473  402  417  5976  5991 CAATGTTCCGACTCCT73  979 915493  441  456  6015  6030 GGAGGAACTTGCTTAA 87  980 915513 470  485  6044  6059 GGCCGGGAGGCATTTG  0  981 915533  500  515  6074 6089 TATTTTGCCGGAGATG 75  982 915553  522  537  6096  6111ACACTCTGGTAAGAGA  0  983 915613  710  725 11928 11943 CACGGTGATGGTTGTT64  984 915633  788  803 12006 12021 GAGCTTGGTGATGTCC 74  985 915653 837  852 12055 12070 CAAAAGCTCTCGAGAG  0  986 915673  892  907 1362813643 GCATCCAAATATCCTC 81  987 915693  973  988 16093 16108TCAGGATCCATCCCTT 10  988 915713 1007 1022 16127 16142 CAGACTCATGTTTGCC 0  989 915733 1082 1097 16202 16217 GCTGAGACGCAGGTGG 64  990 9157531142 1157 N/A N/A CAGTGCTGTAGCGAGC  0  991 915773 1204 1219 19044 19059CTAATGGGTAGCAAGT 72  992 915793 1258 1273 19098 19113 GCAATGGCAGATTCCA57  993 915813 1329 1344 23734 23749 AGGTCACCCACTGCAA 56  994 9158331359 1374 23764 23779 GACACATCAGCACTCG 43  995 915853 1431 1446 2519425209 AGTCCTGCTCAGGTGT 66  996 915873 1525 1540 25288 25303AAGTTCAGGCTGGACC 54  997 915893 1571 1586 25334 25349 GGTGGAGAGCCCCTCA 0  998 915913 1622 1637 25385 25400 CTCGCCTCCTCAAGTG 52  999 9159331660 1675 25423 25438 GATGGGAAACTTTAGC 85 1000 915953 1697 1712 2546025475 GGGTCACTACACAGCA 78 1001 915973 1778 1793 25541 25556CTGCACAGGCGAAAGG 35 1002 915993 1800 1815 25563 25578 ATTAGAGTTAAGTGCT63 1003 916013 1827 1842 25590 25605 ACCAGCTGAATTAACG 66 1004 9160331873 1888 25636 25651 GTCAGTAAGGGACCCT 52 1005 916053 1897 1912 2566025675 GACCATTAATAGGGCC 51 1006 916073 1924 1939 25687 25702TCTAAGAACCTCATGC 55 1007 916093 1967 1982 25730 25745 TACCCCCCATCACAAG15 1008 916113 1990 2005 25753 25768 ACAAGATCACACATGG 72 1009 9161332086 2101 25849 25864 AGACCACCTGACAGGC 79 1010 916152 2112 2127 2587525890 TTAGTAGTCAAGGTTA 84 1011 916172 2177 2192 25940 25955AGGTGAAAAAGGTGTT 88 1012 916192 2285 2300 26048 26063 TTAAGGCTTATTCTAC82 1013 916212 2635 2650 26398 26413 TGAGGTGAATGCCCTG 58 1014 9162322696 2711 26459 26474 TGTGTGCTCCAGTGGA 89 1015 916252 2730 2745 2649326508 GCTGGTCCCTGCAGAA 44 1016 916272 N/A N/A  3328  3343GGGACGCACGAGAGTC  0 1017 916292 N/A N/A  4432  4447 GTCAATAGCTTCACAA 861018 916312 N/A N/A  5281  5296 ATAAATCTTGGGATGC 92 1019 916332 N/A N/A 5577  5592 CACAATGACATCATGG 95 1020 916352 N/A N/A  6170  6185GATAAGCAAACTGCTT 19 1021 916372 N/A N/A  7192  7207 GAGGATGCAACTGGCT 841022 916392 N/A N/A  7644  7659 TCGGACTTCAGGCCCA  0 1023 916552 N/A N/A12208 12223 CCTTTTATTTCCGTTA 97 1024 916572 N/A N/A 12745 12760GCATACTAAAACCACC 85 1025 916592 N/A N/A 13375 13390 GACTTTGCAGGCACCC 921026 916612 N/A N/A 13909 13924 TGACATCCCAGTTCAA 30 1027 916632 N/A N/A14427 14442 TACTTTCCCATCGACA 81 1028 916652 N/A N/A 15207 15222AGGAATATTGCCAGGT 88 1029 916672 N/A N/A 15768 15783 GGTTAGTGTTGGTTTA 921030 916692 N/A N/A 16790 16805 CATTCGATGGAGGTTC 58 1031 916712 N/A N/A17629 17644 GGCGGATTTCCCCACT 11 1032 916732 N/A N/A 18894 18909TAAAATACGCCCGTCC  7 1033 916752 N/A N/A 20183 20198 TCCCTATGTTCTACTT 321034 916771 N/A N/A 20574 20589 ATCAACATCTCTAGCT 46 1035 916791 N/A N/A20811 20826 GGGTAATATTCAGACC 43 1036 916811 N/A N/A 21313 21328TTTACTAGAGACTCTG 69 1037 916831 N/A N/A 22071 22086 GTAGGATAGGACTAGA 451038 916851 N/A N/A 23219 23234 ATAAATGCCTGACCAC 64 1039 916871 N/A N/A23861 23876 TGTTTCTAGAATGTCG 68 1040 916891 N/A N/A 24873 24888GCCTATCAGTTTCCCC  0 1041

TABLE 16Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915354   36   51  2774  2789CTCGGCCAGGGCATTC  0 1042 915374   89  104  2827  2842 TCCGCAGCAGCTCCGC60 1043 915394  137  152  2875  2890 GCGCGGGTTAGGATCT  0 1044 915414 240  255  2978  2993 AGCGGGTCGCCCCGAC 21 1045 915434  337  352  3075 3090 ATACCGGAGAGGACGC 85 1046 915454  374  389  5948  5963AAGATCTGAGAGGACC 24 1047 915474  403  418  5977  5992 CCAATGTTCCGACTCC95 1048 915494  442  457  6016  6031 CGGAGGAACTTGCTTA 93 1049 915514 471  486  6045  6060 TGGCCGGGAGGCATTT  0 1050 915534  501  516  6075 6090 CTATTTTGCCGGAGAT 87 1051 915554  523  538  6097  6112GACACTCTGGTAAGAG 26 1052 915614  711  726 11929 11944 ACACGGTGATGGTTGT46 1053 915634  791  806 12009 12024 ACTGAGCTTGGTGATG 87 1054 915654 838  853 12056 12071 ACAAAAGCTCTCGAGA  0 1055 915674  900  915 1363613651 ACCTGAATGCATCCAA 93 1056 915694  974  989 16094 16109CTCAGGATCCATCCCT 43 1057 915714 1008 1023 16128 16143 CCAGACTCATGTTTGC 0 1058 915734 1083 1098 16203 16218 TGCTGAGACGCAGGTG 50 1059 9157541143 1158 N/A N/A TCAGTGCTGTAGCGAG 42 1060 915774 1208 1223 19048 19063TATCCTAATGGGTAGC 53 1061 915794 1260 1275 19100 19115 TCGCAATGGCAGATTC67 1062 915814 1333 1348 23738 23753 TGTGAGGTCACCCACT  0 1063 9158341360 1375 23765 23780 AGACACATCAGCACTC 24 1064 915854 1432 1447 2519525210 CAGTCCTGCTCAGGTG 54 1065 915874 1526 1541 25289 25304GAAGTTCAGGCTGGAC 75 1066 915894 1572 1587 25335 25350 AGGTGGAGAGCCCCTC 0 1067 915914 1623 1638 25386 25401 ACTCGCCTCCTCAAGT  0 1068 9159341661 1676 25424 25439 AGATGGGAAACTTTAG 84 1069 915954 1724 1739 2548725502 GGCTGGGATCCTCCAC 24 1070 915974 1779 1794 25542 25557GCTGCACAGGCGAAAG 56 1071 915994 1801 1816 25564 25579 TATTAGAGTTAAGTGC75 1072 916014 1828 1843 25591 25606 AACCAGCTGAATTAAC 55 1073 9160341875 1890 25638 25653 CAGTCAGTAAGGGACC 70 1074 916054 1898 1913 2566125676 TGACCATTAATAGGGC 74 1075 916074 1925 1940 25688 25703TTCTAAGAACCTCATG 22 1076 916094 1968 1983 25731 25746 CTACCCCCCATCACAA 0 1077 916114 1992 2007 25755 25770 CCACAAGATCACACAT  0 1078 9161342087 2102 25850 25865 CAGACCACCTGACAGG 78 1079 916153 2113 2128 2587625891 TTTAGTAGTCAAGGTT 93 1080 916173 2178 2193 25941 25956TAGGTGAAAAAGGTGT 89 1081 916193 2306 2321 26069 26084 ACCCAACCGATTTTTT61 1082 916213 2636 2651 26399 26414 CTGAGGTGAATGCCCT 73 1083 9162332697 2712 26460 26475 TTGTGTGCTCCAGTGG 92 1084 916253 2746 2761 2650926524 TCACTGACCATGTGGG 16 1085 916273 N/A N/A  3362  3377CTTCATGCACGGGCGC 37 1086 916293 N/A N/A  4462  4477 GCATAATCTCCTGCCT  01087 916313 N/A N/A  5284  5299 GCCATAAATCTTGGGA 37 1088 916333 N/A N/A 5605  5620 CTTTATTCAATGTGGC 97 1089 916353 N/A N/A  6529  6544TACAACTGCCTGTGTT  0 1090 916373 N/A N/A  7218  7233 AAAGCTTCCGCAAACA 511091 916393 N/A N/A  7657  7672 CTAACATACACCCTCG  0 1092 916553 N/A N/A12225 12240 AGCTTCTGGGACAAGC 10 1093 916573 N/A N/A 12746 12761GGCATACTAAAACCAC 55 1094 916593 N/A N/A 13397 13412 TTGAATGTCACCCTTC 911095 916613 N/A N/A 13914 13929 AGTCATGACATCCCAG 93 1096 916633 N/A N/A14442 14457 TCTCATTGGCACCTGT 86 1097 916653 N/A N/A 15252 15267CCCTATCAGATGCCCT 81 1098 916673 N/A N/A 15799 15814 CATATCTGGTTTCATG  01099 916693 N/A N/A 16842 16857 GACCATAGCACTGTCT  0 1100 916713 N/A N/A17737 17752 ATTAATCTGGTCATAT  0 1101 916733 N/A N/A 18898 18913TCCATAAAATACGCCC 69 1102 916753 N/A N/A 20195 20210 GAAAGATGGAATTCCC 861103 916772 N/A N/A 20604 20619 TACGATCATCATTATT 91 1104 916792 N/A N/A20841 20856 GTATTAGCTCAATATT  0 1105 916812 N/A N/A 21314 21329GTTTACTAGAGACTCT 64 1106 916832 N/A N/A 22080 22095 GTAAAAACTGTAGGAT  01107 916852 N/A N/A 23220 23235 GATAAATGCCTGACCA 29 1108 916872 N/A N/A24011 24026 CCGACGGGAAGTCTTC  0 1109 916892 N/A N/A 24874 24889GGCCTATCAGTTTCCC  0 1110

TABLE 17Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915355   37   52  2775  2790TCTCGGCCAGGGCATT  0 1111 915375   90  105  2828  2843 ATCCGCAGCAGCTCCG52 1112 915395  138  153  2876  2891 GGCGCGGGTTAGGATC  0 1113 915415 241  256  2979  2994 CAGCGGGTCGCCCCGA  8 1114 915435  338  353  3076 3091 GATACCGGAGAGGACG 30 1115 915455  378  393  5952  5967GCACAAGATCTGAGAG 72 1116 915475  405  420  5979  5994 TGCCAATGTTCCGACT 0 1117 915495  443  458  6017  6032 TCGGAGGAACTTGCTT 69 1118 915515 472  487  6046  6061 TTGGCCGGGAGGCATT  9 1119 915535  502  517  6076 6091 CCTATTTTGCCGGAGA 96 1120 915555  524  539  6098  6113AGACACTCTGGTAAGA  2 1121 915615  712  727 11930 11945 GACACGGTGATGGTTG32 1122 915635  792  807 12010 12025 GACTGAGCTTGGTGAT 93 1123 915655 839  854 12057 12072 GACAAAAGCTCTCGAG 40 1124 915675  901  916 1363713652 AACCTGAATGCATCCA 92 1125 915695  975  990 16095 16110CCTCAGGATCCATCCC  0 1126 915715 1011 1026 16131 16146 AATCCAGACTCATGTT67 1127 915735 1088 1103 16208 16223 CAGGATGCTGAGACGC 86 1128 9157551144 1159 N/A N/A CTCAGTGCTGTAGCGA 25 1129 915775 1209 1224 19049 19064TTATCCTAATGGGTAG 23 1130 915795 1261 1276 19101 19116 ATCGCAATGGCAGATT 0 1131 915815 1337 1352 23742 23757 CACCTGTGAGGTCACC 35 1132 9158351361 1376 23766 23781 CAGACACATCAGCACT 54 1133 915855 1433 1448 2519625211 CCAGTCCTGCTCAGGT 23 1134 915875 1530 1545 25293 25308AGAAGAAGTTCAGGCT 81 1135 915895 1574 1589 25337 25352 AAAGGTGGAGAGCCCC76 1136 915915 1624 1639 25387 25402 GACTCGCCTCCTCAAG 75 1137 9159351674 1689 25437 25452 GGTAGCTGCACAAAGA 76 1138 915955 1726 1741 2548925504 GAGGCTGGGATCCTCC  0 1139 915975 1780 1795 25543 25558CGCTGCACAGGCGAAA  0 1140 915995 1805 1820 25568 25583 GATGTATTAGAGTTAA82 1141 916015 1829 1844 25592 25607 CAACCAGCTGAATTAA 59 1142 9160351876 1891 25639 25654 ACAGTCAGTAAGGGAC 81 1143 916055 1899 1914 2566225677 CTGACCATTAATAGGG 49 1144 916075 1929 1944 25692 25707GTCATTCTAAGAACCT 81 1145 916095 1969 1984 25732 25747 CCTACCCCCCATCACA21 1146 916115 1995 2010 25758 25773 ACCCCACAAGATCACA  0 1147 9161352088 2103 25851 25866 GCAGACCACCTGACAG 44 1148 916154 2131 2146 2589425909 CCCCGCCATGGAGACG 68 1149 916174 2180 2195 25943 25958GTTAGGTGAAAAAGGT 90 1150 916194 2308 2323 26071 26086 GCACCCAACCGATTTT83 1151 916214 2637 2652 26400 26415 GCTGAGGTGAATGCCC 52 1152 9162342698 2713 26461 26476 GTTGTGTGCTCCAGTG 88 1153 916254 2747 2762 2651026525 CTCACTGACCATGTGG 13 1154 916274 N/A N/A  3524  3539GCAAATCGGCCCCTCG  3 1155 916294 N/A N/A  4463  4478 GGCATAATCTCCTGCC  01156 916314 N/A N/A  5324  5339 TGGCATGCAAGACCAC  0 1157 916334 N/A N/A 5606  5621 ACTTTATTCAATGTGG 95 1158 916354 N/A N/A  6556  6571GTTTATGTCACTCTGG 68 1159 916374 N/A N/A  7245  7260 GAACAGACAAGTGCTG 381160 916394 N/A N/A  7658  7673 ACTAACATACACCCTC 31 1161 916554 N/A N/A12249 12264 ATAATCAGGGTGGTGC  0 1162 916574 N/A N/A 12747 12762AGGCATACTAAAACCA 47 1163 916594 N/A N/A 13500 13515 GAATCATGCAAGCTCT 501164 916614 N/A N/A 13996 14011 TAAACTAAGGGTCACA 37 1165 916634 N/A N/A14497 14512 ATCCATCCTGCATGAG 76 1166 916654 N/A N/A 15254 15269GGCCCTATCAGATGCC  0 1167 916674 N/A N/A 15802 15817 CTACATATCTGGTTTC  01168 916694 N/A N/A 16844 16859 TGGACCATAGCACTGT 60 1169 916714 N/A N/A17738 17753 TATTAATCTGGTCATA 18 1170 916734 N/A N/A 18926 18941CCACTTTACTCTGTTG 64 1171 916754 N/A N/A 20210 20225 AACTATGCCTAGAACG 431172 916773 N/A N/A 20606 20621 TTTACGATCATCATTA 77 1173 916793 N/A N/A20842 20857 TGTATTAGCTCAATAT  0 1174 916813 N/A N/A 21319 21334TGGGAGTTTACTAGAG 66 1175 916833 N/A N/A 22118 22133 AGAGAGTACTCTTGGA 111176 916853 N/A N/A 23222 23237 CTGATAAATGCCTGAC 78 1177 916873 N/A N/A24038 24053 ATCAATGCTGCACTCA 88 1178 916893 N/A N/A 24889 24904ACGAATCCCTGGAGGG  0 1179

TABLE 18Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915356   38   53  2776  2791GTCTCGGCCAGGGCAT  0 1180 915376   93  108  2831  2846 CTGATCCGCAGCAGCT28 1181 915396  165  180  2903  2918 CGTACATGGCGGCGGC  0 1182 915416 242  257  2980  2995 GCAGCGGGTCGCCCCG  0 1183 915436  339  354  3077 3092 GGATACCGGAGAGGAC 64 1184 915456  379  394  5953  5968CGCACAAGATCTGAGA 79 1185 915476  406  421  5980  5995 ATGCCAATGTTCCGAC83 1186 915496  444  459  6018  6033 GTCGGAGGAACTTGCT 35 1187 915516 473  488  6047  6062 ATTGGCCGGGAGGCAT  0 1188 915536  503  518  6077 6092 GCCTATTTTGCCGGAG 77 1189 915556  525  540  6099  6114CAGACACTCTGGTAAG 62 1190 915616  730  745 11948 11963 TACTCCCCATAGAAGG 8 1191 915636  794  809 12012 12027 TAGACTGAGCTTGGTG 90 1192 915656 840  855 12058 12073 GGACAAAAGCTCTCGA 60 1193 915676  902  917 1363813653 GAACCTGAATGCATCC 72 1194 915696  978  993 16098 16113CGACCTCAGGATCCAT  0 1195 915716 1012 1027 16132 16147 GAATCCAGACTCATGT 0 1196 915736 1089 1104 16209 16224 GCAGGATGCTGAGACG 55 1197 9157561145 1160 N/A N/A ACTCAGTGCTGTAGCG 12 1198 915776 1210 1225 19050 19065ATTATCCTAATGGGTA 28 1199 915796 1262 1277 19102 19117 AATCGCAATGGCAGAT 0 1200 915816 1339 1354 23744 23759 AACACCTGTGAGGTCA 56 1201 9158361365 1380 23770 23785 GGAGCAGACACATCAG 53 1202 915856 1434 1449 2519725212 GCCAGTCCTGCTCAGG 21 1203 915876 1531 1546 25294 25309AAGAAGAAGTTCAGGC 85 1204 915896 1575 1590 25338 25353 GAAAGGTGGAGAGCCC78 1205 915916 1626 1641 25389 25404 TAGACTCGCCTCCTCA 32 1206 9159361676 1691 25439 25454 GAGGTAGCTGCACAAA 91 1207 915956 1737 1752 2550025515 AACTCAGCTCAGAGGC 46 1208 915976 1781 1796 25544 25559CCGCTGCACAGGCGAA  0 1209 915996 1807 1822 25570 25585 CTGATGTATTAGAGTT93 1210 916016 1830 1845 25593 25608 CCAACCAGCTGAATTA 21 1211 9160361877 1892 25640 25655 AACAGTCAGTAAGGGA 82 1212 916056 1900 1915 2566325678 TCTGACCATTAATAGG 13 1213 916076 1930 1945 25693 25708TGTCATTCTAAGAACC 40 1214 916096 1970 1985 25733 25748 GCCTACCCCCCATCAC18 1215 916116 1996 2011 25759 25774 CACCCCACAAGATCAC 50 1216 9161362089 2104 25852 25867 TGCAGACCACCTGACA 58 1217 916155 2132 2147 2589525910 CCCCCGCCATGGAGAC 33 1218 916175 2224 2239 25987 26002CGCTTCCTTACATTTT 89 1219 916195 2309 2324 26072 26087 TGCACCCAACCGATTT64 1220 916215 2638 2653 26401 26416 GGCTGAGGTGAATGCC  0 1221 9162352699 2714 26462 26477 AGTTGTGTGCTCCAGT 85 1222 916255 2748 2763 2651126526 ACTCACTGACCATGTG  0 1223 916275 N/A N/A  3555  3570GGCCAAAGCCCCACTC  0 1224 916295 N/A N/A  4464  4479 GGGCATAATCTCCTGC  01225 916315 N/A N/A  5342  5357 GGCTGATCTGCACTCT 84 1226 916335 N/A N/A 5626  5641 TAATTCTACCTGTGTC 92 1227 916355 N/A N/A  6557  6572AGTTTATGTCACTCTG 27 1228 916375 N/A N/A  7321  7336 ACACTTTGCGAAGCAC 271229 916395 N/A N/A  7660  7675 GAACTAACATACACCC  1 1230 916555 N/A N/A12252 12267 CCCATAATCAGGGTGG  0 1231 916575 N/A N/A 12758 12773GTAGAGTGGTAAGGCA 95 1232 916595 N/A N/A 13502 13517 AAGAATCATGCAAGCT 341233 916615 N/A N/A 13997 14012 TTAAACTAAGGGTCAC 65 1234 916635 N/A N/A14549 14564 TTAATGTGGATTCACG 76 1235 916655 N/A N/A 15295 15310CCAAGATAACCTCACA 64 1236 916675 N/A N/A 15806 15821 CCATCTACATATCTGG 261237 916695 N/A N/A 16854 16869 CACAATCATTTGGACC 72 1238 916715 N/A N/A17739 17754 GTATTAATCTGGTCAT 87 1239 916735 N/A N/A 19113 19128CACCTCTGGACAATCG 29 1240 916755 N/A N/A 20212 20227 CAAACTATGCCTAGAA 701241 916774 N/A N/A 20608 20623 ATTTTACGATCATCAT 91 1242 916794 N/A N/A20846 20861 TGCCTGTATTAGCTCA 90 1243 916814 N/A N/A 21345 21360CACATAAAGTCAAACG 87 1244 916834 N/A N/A 22124 22139 AGAACAAGAGAGTACT  31245 916854 N/A N/A 23250 23265 CACATAAAGGACCCCC 54 1246 916874 N/A N/A24126 24141 CGCTATCTGACACTCC 87 1247 916894 N/A N/A 24896 24911TCCACCAACGAATCCC 50 1248

TABLE 19Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915357   39   54  2777  2792TGTCTCGGCCAGGGCA  0 1249 915377   94  109  2832  2847 CCTGATCCGCAGCAGC 0 1250 915397  182  197  2920  2935 CCAGCCGCGCTCTGCG  0 1251 915417 243  258  2981  2996 GGCAGCGGGTCGCCCC  0 1252 915437  341  356  3079 3094 CGGGATACCGGAGAGG 57 1253 915457  380  395  5954  5969CCGCACAAGATCTGAG 71 1254 915477  407  422  5981  5996 GATGCCAATGTTCCGA93 1255 915497  445  460  6019  6034 TGTCGGAGGAACTTGC 85 1256 915517 474  489  6048  6063 CATTGGCCGGGAGGCA  0 1257 915537  504  519  6078 6093 TGCCTATTTTGCCGGA 44 1258 915557  526  541  6100  6115TCAGACACTCTGGTAA 26 1259 915617  732  747 11950 11965 CGTACTCCCCATAGAA66 1260 915637  796  811 12014 12029 CGTAGACTGAGCTTGG 98 1261 915657 857  872 12075 12090 CACCTTGAGATCCGGG  0 1262 915677  903  918 1363913654 AGAACCTGAATGCATC 78 1263 915697  979  994 16099 16114GCGACCTCAGGATCCA  1 1264 915717 1031 1046 16151 16166 GGCAGCCGACTCCGGG19 1265 915737 1109 1124 16229 16244 CAGGATGCTCTCATCC  0 1266 9157571146 1161 N/A N/A CACTCAGTGCTGTAGC 33 1267 915777 1214 1229 19054 19069AGACATTATCCTAATG 42 1268 915797 1263 1278 19103 19118 CAATCGCAATGGCAGA49 1269 915817 1340 1355 23745 23760 GAACACCTGTGAGGTC 44 1270 9158371398 1413 25161 25176 GGCTGCTCACTGGCAT 18 1271 915857 1435 1450 2519825213 GGCCAGTCCTGCTCAG  0 1272 915877 1534 1549 25297 25312CCCAAGAAGAAGTTCA 76 1273 915897 1576 1591 25339 25354 GGAAAGGTGGAGAGCC24 1274 915917 1627 1642 25390 25405 CTAGACTCGCCTCCTC 77 1275 9159371679 1694 25442 25457 GCGGAGGTAGCTGCAC 16 1276 915957 1738 1753 2550125516 CAACTCAGCTCAGAGG 61 1277 915977 1782 1797 25545 25560ACCGCTGCACAGGCGA 34 1278 915997 1809 1824 25572 25587 TGCTGATGTATTAGAG83 1279 916017 1831 1846 25594 25609 CCCAACCAGCTGAATT 42 1280 9160371878 1893 25641 25656 AAACAGTCAGTAAGGG 92 1281 916057 1901 1916 2566425679 GTCTGACCATTAATAG 64 1282 916077 1931 1946 25694 25709CTGTCATTCTAAGAAC 41 1283 916097 1971 1986 25734 25749 AGCCTACCCCCCATCA 0 1284 916117 1997 2012 25760 25775 CCACCCCACAAGATCA  0 1285 9161372090 2105 25853 25868 TTGCAGACCACCTGAC 65 1286 916156 2133 2148 2589625911 ACCCCCGCCATGGAGA 54 1287 916176 2225 2240 25988 26003ACGCTTCCTTACATTT 84 1288 916196 2310 2325 26073 26088 CTGCACCCAACCGATT58 1289 916216 2639 2654 26402 26417 GGGCTGAGGTGAATGC 46 1290 9162362700 2715 26463 26478 AAGTTGTGTGCTCCAG 86 1291 916256 2751 2766 2651426529 GAAACTCACTGACCAT 41 1292 916276 N/A N/A  4068  4083GGAAACAACTTTCCTC  0 1293 916296 N/A N/A  4730  4745 GATCATGTGGCGGTCT 681294 916316 N/A N/A  5364  5379 CACTTACTGGCCTGGC 30 1295 916336 N/A N/A 5645  5660 ATATTGGGCTCAATGA 89 1296 916356 N/A N/A  6575  6590ATCACTGGAGGTGTAC  0 1297 916376 N/A N/A  7328  7343 CAGGATCACACTTTGC 171298 916396 N/A N/A  7661  7676 GGAACTAACATACACC  0 1299 916556 N/A N/A12272 12287 GTATATGTTCCCAGGT 81 1300 916576 N/A N/A 12788 12803GTGTACATGGTCTGCA 94 1301 916596 N/A N/A 13529 13544 ATCATTGGAAGACCGC 891302 916616 N/A N/A 13998 14013 GTTAAACTAAGGGTCA 85 1303 916636 N/A N/A14550 14565 CTTAATGTGGATTCAC 91 1304 916656 N/A N/A 15351 15366TCCAACTTCAGGCTGA 74 1305 916676 N/A N/A 15819 15834 AGCTTTGTGGGCTCCA 691306 916696 N/A N/A 16982 16997 GTTTAATAAGGGCACC 63 1307 916716 N/A N/A17740 17755 CGTATTAATCTGGTCA 93 1308 916736 N/A N/A 19126 19141CACCTAAAATGCTCAC 17 1309 916756 N/A N/A 20213 20228 ACAAACTATGCCTAGA 581310 916775 N/A N/A 20609 20624 AATTTTACGATCATCA 78 1311 916795 N/A N/A20927 20942 GACAGATCAGCACTCG 80 1312 916815 N/A N/A 21407 21422CAATTCTAGACATGGC 88 1313 916835 N/A N/A 22338 22353 TGCACCTACCCTTTTC 391314 916855 N/A N/A 23251 23266 ACACATAAAGGACCCC 48 1315 916875 N/A N/A24241 24256 GCATTACCAGGCACCT 61 1316 916895 N/A N/A 24912 24927GACATCACAGGTGTTG  5 1317

TABLE 20Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915358   40   55  2778  2793GTGTCTCGGCCAGGGC  0 1318 915378   96  111  2834  2849 GTCCTGATCCGCAGCA 0 1319 915398  184  199  2922  2937 CTCCAGCCGCGCTCTG 14 1320 915418 244  259  2982  2997 AGGCAGCGGGTCGCCC  0 1321 915438  342  357  3080 3095 GCGGGATACCGGAGAG 44 1322 915458  381  396  5955  5970TCCGCACAAGATCTGA 41 1323 915478  408  423  5982  5997 AGATGCCAATGTTCCG95 1324 915498  446  461  6020  6035 CTGTCGGAGGAACTTG 40 1325 915518 475  490  6049  6064 ACATTGGCCGGGAGGC  5 1326 915538  505  520  6079 6094 ATGCCTATTTTGCCGG 61 1327 915558  527  542  6101  6116ATCAGACACTCTGGTA  0 1328 915618  748  763 11966 11981 ACTTTAGGGCAGATGT87 1329 915638  818  833 12036 12051 GTAGAGGTTCCCTGTG 87 1330 915658 859  874 N/A N/A AGCACCTTGAGATCCG  0 1331 915678  926  941 N/A N/AGTTGCAGATGCCCTTC 24 1332 915698  980  995 16100 16115 GGCGACCTCAGGATCC 0 1333 915718 1032 1047 16152 16167 AGGCAGCCGACTCCGG  8 1334 9157381114 1129 16234 16249 GTGTCCAGGATGCTCT 41 1335 915758 1147 1162 N/A N/ATCACTCAGTGCTGTAG 59 1336 915778 1217 1232 19057 19072 ATAAGACATTATCCTA85 1337 915798 1264 1279 19104 19119 ACAATCGCAATGGCAG 66 1338 9158181342 1357 23747 23762 GTGAACACCTGTGAGG 58 1339 915838 1400 1415 2516325178 TTGGCTGCTCACTGGC 79 1340 915858 1436 1451 25199 25214GGGCCAGTCCTGCTCA  0 1341 915878 1535 1550 25298 25313 GCCCAAGAAGAAGTTC54 1342 915898 1590 1605 25353 25368 CTAGTGAAAAACTGGG 34 1343 9159181628 1643 25391 25406 GCTAGACTCGCCTCCT 33 1344 915938 1680 1695 2544325458 TGCGGAGGTAGCTGCA  0 1345 915958 1756 1771 25519 25534CCTAGCTTTTCATAAA  0 1346 915978 1783 1798 25546 25561 GACCGCTGCACAGGCG24 1347 915998 1810 1825 25573 25588 ATGCTGATGTATTAGA 86 1348 9160181832 1847 25595 25610 TCCCAACCAGCTGAAT  3 1349 916038 1879 1894 2564225657 GAAACAGTCAGTAAGG 64 1350 916058 1902 1917 25665 25680AGTCTGACCATTAATA 86 1351 916078 1933 1948 25696 25711 ACCTGTCATTCTAAGA18 1352 916098 1972 1987 25735 25750 CAGCCTACCCCCCATC 41 1353 9161181999 2014 25762 25777 CTCCACCCCACAAGAT  0 1354 916138 2092 2107 2585525870 CTTTGCAGACCACCTG 65 1355 916157 2134 2149 25897 25912TACCCCCGCCATGGAG 57 1356 916177 2237 2252 26000 26015 CAACAGGTAACAACGC88 1357 916197 2579 2594 26342 26357 GTCAGACTTTCACTCA 81 1358 9162172659 2674 26422 26437 GTGCTTGGCTCCTGCC 43 1359 916237 2701 2716 2646426479 CAAGTTGTGTGCTCCA 73 1360 916257 2769 2784 26532 26547CATCGCCACACATGGG 61 1361 916277 N/A N/A  4105  4120 AGGAAGGGTCCCAAAC  01362 916297 N/A N/A  4731  4746 TGATCATGTGGCGGTC 80 1363 916317 N/A N/A 5391  5406 TGCTATCAGGTGCAGG 60 1364 916337 N/A N/A  5646  5661TATATTGGGCTCAATG 71 1365 916357 N/A N/A  6594  6609 GTTTACAAACATGGAC 261366 916377 N/A N/A  7464  7479 TCATTAGCATCACCGG 33 1367 916397 N/A N/A 7662  7677 GGGAACTAACATACAC  0 1368 916557 N/A N/A 12274 12289GGGTATATGTTCCCAG  0 1369 916577 N/A N/A 12830 12845 TGCATAGCCTTCTTTC 841370 916597 N/A N/A 13530 13545 CATCATTGGAAGACCG 62 1371 916617 N/A N/A14016 14031 TCTTTAACTTCGGCCC 70 1372 916637 N/A N/A 14551 14566TCTTAATGTGGATTCA 88 1373 916657 N/A N/A 15388 15403 TCAGACAACCACAGCT 661374 916677 N/A N/A 15852 15867 TAAAGCAGGACACACG 74 1375 916697 N/A N/A17077 17092 AGACATGTTGGTGTCT  0 1376 916717 N/A N/A 17788 17803CCCCAGTCTTTTATTC  0 1377 916737 N/A N/A 19140 19155 GGAAGACACGGAGCCA 201378 916757 N/A N/A 20240 20255 CCTAACTGCTGGCTCT 85 1379 916776 N/A N/A20610 20625 TAATTTTACGATCATC 76 1380 916796 N/A N/A 20939 20954CTCTTTGTAGCAGACA 90 1381 916816 N/A N/A 21439 21454 CAATATACTGAGAGGA 921382 916836 N/A N/A 22392 22407 GTAGACATCCTTCCCG 65 1383 916856 N/A N/A23252 23267 GACACATAAAGGACCC 59 1384 916876 N/A N/A 24242 24257TGCATTACCAGGCACC 37 1385 916896 N/A N/A 24913 24928 GGACATCACAGGTGTT 191386

TABLE 21Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915359   41   56  2779  2794AGTGTCTCGGCCAGGG  0 1387 915379   97  112  2835  2850 GGTCCTGATCCGCAGC 0 1388 915399  185  200  2923  2938 GCTCCAGCCGCGCTCT 26 1389 915419 245  260  2983  2998 CAGGCAGCGGGTCGCC  0 1390 915439  343  358  3081 3096 AGCGGGATACCGGAGA 69 1391 915459  382  397  5956  5971TTCCGCACAAGATCTG 71 1392 915479  409  424  5983  5998 AAGATGCCAATGTTCC93 1393 915499  448  463  6022  6037 CCCTGTCGGAGGAACT 30 1394 915519 477  492  6051  6066 GGACATTGGCCGGGAG 88 1395 915539  506  521  6080 6095 GATGCCTATTTTGCCG 60 1396 915559  529  544  6103  6118CCATCAGACACTCTGG 30 1397 915579  595  610  7825  7840 CAGGAACATACCAAGG98 1398 915599  674  689 11892 11907 GTTGTCACTCACTCCT 98 1399 915619 749  764 11967 11982 GACTTTAGGGCAGATG 96 1400 915639  819  834 1203712052 GGTAGAGGTTCCCTGT 92 1401 915659  860  875 N/A N/A CAGCACCTTGAGATCC 0 1402 915679  928  943 N/A N/A CTGTTGCAGATGCCCT 58 1403 915699  981 996 16101 16116 TGGCGACCTCAGGATC 40 1404 915719 1033 1048 16153 16168AAGGCAGCCGACTCCG  0 1405 915739 1115 1130 16235 16250 GGTGTCCAGGATGCTC40 1406 915759 1148 1163 N/A N/A TTCACTCAGTGCTGTA 29 1407 915779 12191234 19059 19074 ACATAAGACATTATCC 86 1408 915799 1268 1283 19108 19123CTGGACAATCGCAATG 42 1409 915819 1344 1359 23749 23764 GAGTGAACACCTGTGA81 1410 915839 1401 1416 25164 25179 GTTGGCTGCTCACTGG 85 1411 9158591437 1452 25200 25215 AGGGCCAGTCCTGCTC  0 1412 915879 1538 1553 2530125316 ATTGCCCAAGAAGAAG 54 1413 915899 1591 1606 25354 25369TCTAGTGAAAAACTGG  0 1414 915919 1629 1644 25392 25407 TGCTAGACTCGCCTCC72 1415 915939 1681 1696 25444 25459 ATGCGGAGGTAGCTGC 39 1416 9159591764 1779 25527 25542 GGTTGCTTCCTAGCTT 87 1417 915979 1784 1799 2554725562 GGACCGCTGCACAGGC  0 1418 915999 1811 1826 25574 25589CATGCTGATGTATTAG 35 1419 916019 1833 1848 25596 25611 TTCCCAACCAGCTGAA 0 1420 916039 1880 1895 25643 25658 CGAAACAGTCAGTAAG 80 1421 9160591905 1920 25668 25683 AACAGTCTGACCATTA 85 1422 916079 1934 1949 2569725712 CACCTGTCATTCTAAG 45 1423 916099 1973 1988 25736 25751CCAGCCTACCCCCCAT 53 1424 916119 2022 2037 25785 25800 GTGGGATCATGCTATT76 1425 916139 2093 2108 25856 25871 TCTTTGCAGACCACCT 85 1426 9161582135 2150 25898 25913 TTACCCCCGCCATGGA  0 1427 916178 2238 2253 2600126016 TCAACAGGTAACAACG 87 1428 916198 2620 2635 26383 26398GCACACTAGATTATTT 66 1429 916218 2673 2688 26436 26451 CGGAAGCTCCTGCTGT27 1430 916238 2702 2717 26465 26480 TCAAGTTGTGTGCTCC 91 1431 9162582770 2785 26533 26548 TCATCGCCACACATGG 49 1432 916278 N/A N/A  4211 4226 TCATTTCCAGGAGTAC 75 1433 916298 N/A N/A  4735  4750CAAATGATCATGTGGC 93 1434 916318 N/A N/A  5394  5409 TAATGCTATCAGGTGC 951435 916338 N/A N/A  5648  5663 GATATATTGGGCTCAA 97 1436 916358 N/A N/A 6596  6611 GGGTTTACAAACATGG 75 1437 916378 N/A N/A  7465  7480TTCATTAGCATCACCG 78 1438 916398 N/A N/A  7686  7701 GTTAATCCATGGGTCA 491439 916418 N/A N/A  8992  9007 AGCCTAAACTTCCTCC 63 1440 916438 N/A N/A 9318  9333 AGAAGAGCCGCCCTGC 77 1441 916458 N/A N/A  9795  9810GCAAGACTAGCAAGTG 85 1442 916478 N/A N/A 10301 10316 AGCATGCGGTATGTAC 671443 916498 N/A N/A 10849 10864 CACACAATTTCTAGGG 82 1444 916518 N/A N/A11346 11361 TTGACAATTAGAACCA 96 1445 916538 N/A N/A 11711 11726ACAAATCCTTACCGAG 54 1446 916558 N/A N/A 12285 12300 GTTTTAGGTCTGGGTA 941447 916578 N/A N/A 12831 12846 TTGCATAGCCTTCTTT 93 1448 916598 N/A N/A13660 13675 CATACATACCCTTCTC  9 1449 916618 N/A N/A 14025 14040CGCAGAAACTCTTTAA 89 1450 916638 N/A N/A 14552 14567 GTCTTAATGTGGATTC 931451 916658 N/A N/A 15421 15436 AGCATTGGCACACTGG 70 1452 916678 N/A N/A15857 15872 GGCTTTAAAGCAGGAC 62 1453 916698 N/A N/A 17079 17094GCAGACATGTTGGTGT  2 1454 916718 N/A N/A 17839 17854 TACAAGCTGGTCCTTG  01455 916738 N/A N/A 19211 19226 GACAATCCAGGTCCCA 70 1456 916758 N/A N/A20285 20300 GAGGAAGCCCAATCAA 81 1457 916777 N/A N/A 20611 20626CTAATTTTACGATCAT 81 1458 916797 N/A N/A 20984 20999 TTAAACTGCCAAGTCC 831459 916817 N/A N/A 21440 21455 CCAATATACTGAGAGG 96 1460 916837 N/A N/A22406 22421 GGTAGCACCGCCAAGT  0 1461 916857 N/A N/A 23301 23316CACCATGGAGAGGTCT  0 1462 916877 N/A N/A 24243 24258 TTGCATTACCAGGCAC 171463 916897 N/A N/A 24934 24949 GCTACCTGGACACCTC 47 1464

TABLE 22Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 841947 2094 2109 25857 25872ATCTTTGCAGACCACC 89 1464 912986 N/A N/A 20288 20303 TCAGAGGAAGCCCAAT 92 254 20318 20333 915360   42   57  2780  2795 CAGTGTCTCGGCCAGG  0 1466915380   98  113  2836  2851 GGGTCCTGATCCGCAG  0 1467 915400  186  201 2924  2939 AGCTCCAGCCGCGCTC  0 1468 915420  246  261  2984  2999TCAGGCAGCGGGTCGC 78 1469 915440  344  359  3082  3097 CAGCGGGATACCGGAG72 1470 915460  383  398  5957  5972 CTTCCGCACAAGATCT  0 1471 915480 411  426  5985  6000 GGAAGATGCCAATGTT 94 1472 915500  449  464  6023 6038 ACCCTGTCGGAGGAAC 40 1473 915520  480  495  6054  6069GGTGGACATTGGCCGG 38 1474 915540  507  522  6081  6096 AGATGCCTATTTTGCC76 1475 915560  556  571  6130  6145 CGAAAGTCAGACACCA 69 1476 915620 750  765 11968 11983 TGACTTTAGGGCAGAT 89 1477 915640  821  836 1203912054 AAGGTAGAGGTTCCCT 10 1478 915660  875  890 13611 13626AAGGCATATCTCTCCC 47 1479 915680  929  944 N/A N/A CCTGTTGCAGATGCCC 221480 915700  982  997 16102 16117 ATGGCGACCTCAGGAT 58 1481 915720 10341049 16154 16169 CAAGGCAGCCGACTCC 63 1482 915740 1121 1136 16241 16256CGAGAGGGTGTCCAGG  0 1483 915760 1149 1164 N/A N/A CTTCACTCAGTGCTGT 131484 915780 1226 1241 19066 19081 CAGCATTACATAAGAC 94 1485 915800 12701285 19110 19125 CTCTGGACAATCGCAA 55 1486 915820 1345 1360 23750 23765CGAGTGAACACCTGTG 83 1487 915840 1402 1417 25165 25180 TGTTGGCTGCTCACTG77 1488 915860 1470 1485 25233 25248 CTGGACAGCCCTTGGG 29 1489 9158801539 1554 25302 25317 TATTGCCCAAGAAGAA 18 1490 915900 1598 1613 2536125376 ACTCTTCTCTAGTGAA 67 1491 915920 1630 1645 25393 25408CTGCTAGACTCGCCTC 88 1492 915940 1682 1697 25445 25460 AATGCGGAGGTAGCTG 0 1493 915960 1765 1780 25528 25543 AGGTTGCTTCCTAGCT 55 1494 9159801785 1800 25548 25563 TGGACCGCTGCACAGG 82 1495 916000 1812 1827 2557525590 GCATGCTGATGTATTA 52 1496 916020 1837 1852 25600 25615TCATTTCCCAACCAGC 94 1497 916040 1881 1896 25644 25659 ACGAAACAGTCAGTAA79 1498 916060 1907 1922 25670 25685 GGAACAGTCTGACCAT 22 1499 9160801936 1951 25699 25714 AACACCTGTCATTCTA 71 1500 916100 1974 1989 2573725752 GCCAGCCTACCCCCCA 23 1501 916120 2023 2038 25786 25801AGTGGGATCATGCTAT  0 1502 916159 2136 2151 25899 25914 GTTACCCCCGCCATGG47 1503 916179 2239 2254 26002 26017 TTCAACAGGTAACAAC 84 1504 9161992621 2636 26384 26399 TGCACACTAGATTATT  0 1505 916219 2674 2689 2643726452 GCGGAAGCTCCTGCTG  6 1506 916239 2704 2719 26467 26482GTTCAAGTTGTGTGCT 85 1507 916259 2771 2786 26534 26549 CTCATCGCCACACATG85 1508 916279 N/A N/A  4218  4233 CGGAATCTCATTTCCA  0 1509 916299 N/AN/A  4736  4751 GCAAATGATCATGTGG 93 1510 916319 N/A N/A  5396  5411CTTAATGCTATCAGGT 83 1511 916339 N/A N/A  5649  5664 GGATATATTGGGCTCA 961512 916359 N/A N/A  6597  6612 AGGGTTTACAAACATG 32 1513 916379 N/A N/A 7466  7481 ATTCATTAGCATCACC 52 1514 916399 N/A N/A  7687  7702GGTTAATCCATGGGTC  0 1515 916559 N/A N/A 12286 12301 AGTTTTAGGTCTGGGT 891516 916579 N/A N/A 12833 12848 CATTGCATAGCCTTCT 96 1517 916599 N/A N/A13661 13676 CCATACATACCCTTCT 21 1518 916619 N/A N/A 14077 14092ACCCACACCTGACTGG 16 1519 916639 N/A N/A 14572 14587 CGCTCCTACTTATCCC 961520 916659 N/A N/A 15427 15442 TCTTACAGCATTGGCA 38 1521 916679 N/A N/A15973 15988 CATCTACCAAACTGCA 73 1522 916699 N/A N/A 17135 17150AACAAACATCGATTTT 47 1523 916719 N/A N/A 17844 17859 AGCTTTACAAGCTGGT  01524 916739 N/A N/A 19213 19228 ACGACAATCCAGGTCC 14 1525 916778 N/A N/A20612 20627 TCTAATTTTACGATCA 92 1526 916798 N/A N/A 20985 21000ATTAAACTGCCAAGTC 72 1527 916818 N/A N/A 21441 21456 ACCAATATACTGAGAG 921528 916838 N/A N/A 22409 22424 AGCGGTAGCACCGCCA  0 1529 916858 N/A N/A23323 23338 TCACATGTGAGCCCAG 46 1530 916878 N/A N/A 24271 24286GTACAACAGAGGGTGG 19 1531 916898 N/A N/A 24978 24993 GGCTGATGTCACCACC 321532

TABLE 23Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915361   43   58  2781  2796TCAGTGTCTCGGCCAG  0 1533 915381  105  120  2843  2858 TCGGCTCGGGTCCTGA 0 1534 915401  188  203  2926  2941 CAAGCTCCAGCCGCGC 22 1535 915421 247  262  2985  3000 CTCAGGCAGCGGGTCG 48 1536 915441  345  360  3083 3098 CCAGCGGGATACCGGA  0 1537 915461  384  399  5958  5973CCTTCCGCACAAGATC 59 1538 915481  414  429  5988  6003 GATGGAAGATGCCAAT83 1539 915501  450  465  6024  6039 GACCCTGTCGGAGGAA 26 1540 915521 482  497  6056  6071 CTGGTGGACATTGGCC  0 1541 915541  508  523  6082 6097 GAGATGCCTATTTTGC 92 1542 915561  557  572  6131  6146CCGAAAGTCAGACACC  0 1543 915601  691  706 11909 11924 GCATCAATGAAGGGTA91 1544 915621  751  766 11969 11984 TTGACTTTAGGGCAGA 85 1545 915641 822  837 12040 12055 GAAGGTAGAGGTTCCC 74 1546 915661  876  891 1361213627 GAAGGCATATCTCTCC 32 1547 915681  930  945 16050 16065GCCTGTTGCAGATGCC  0 1548 915701  983  998 16103 16118 CATGGCGACCTCAGGA 0 1549 915721 1035 1050 16155 16170 CCAAGGCAGCCGACTC 79 1550 9157411122 1137 16242 16257 GCGAGAGGGTGTCCAG  0 1551 915761 1150 1165 1899019005 TCTTCACTCAGTGCTG 41 1552 915781 1227 1242 19067 19082GCAGCATTACATAAGA 75 1553 915801 1273 1288 N/A N/A AGTCTCTGGACAATCG  01554 915821 1346 1361 23751 23766 TCGAGTGAACACCTGT 52 1555 915841 14031418 25166 25181 CTGTTGGCTGCTCACT 80 1556 915861 1471 1486 25234 25249GCTGGACAGCCCTTGG  0 1557 915881 1540 1555 25303 25318 TTATTGCCCAAGAAGA75 1558 915901 1599 1614 25362 25377 GACTCTTCTCTAGTGA 67 1559 9159211631 1646 25394 25409 TCTGCTAGACTCGCCT 50 1560 915941 1683 1698 2544625461 CAATGCGGAGGTAGCT 39 1561 915961 1766 1781 25529 25544AAGGTTGCTTCCTAGC 71 1562 915981 1786 1801 25549 25564 CTGGACCGCTGCACAG 0 1563 916001 1813 1828 25576 25591 CGCATGCTGATGTATT 73 1564 9160211840 1855 25603 25618 GTGTCATTTCCCAACC 61 1565 916041 1882 1897 2564525660 CACGAAACAGTCAGTA 34 1566 916061 1910 1925 25673 25688GCTGGAACAGTCTGAC 82 1567 916081 1942 1957 25705 25720 CATCCAAACACCTGTC69 1568 916101 1975 1990 25738 25753 GGCCAGCCTACCCCCC  2 1569 9161212024 2039 25787 25802 AAGTGGGATCATGCTA 26 1570 916140 2095 2110 2585825873 CATCTTTGCAGACCAC 92 1571 916160 2137 2152 25900 25915TGTTACCCCCGCCATG 51 1572 916180 2257 2272 26020 26035 GATTCACATAATACAA87 1573 916200 2622 2637 26385 26400 CTGCACACTAGATTAT  0 1574 9162202675 2690 26438 26453 GGCGGAAGCTCCTGCT  0 1575 916240 2705 2720 2646826483 GGTTCAAGTTGTGTGC 69 1576 916260 2772 2787 26535 26550TCTCATCGCCACACAT 72 1577 916280 N/A N/A  4220  4235 TACGGAATCTCATTTC 801578 916300 N/A N/A  4791  4806 GGCCACCTTGGGATAC 17 1579 916320 N/A N/A 5398  5413 GCCTTAATGCTATCAG 67 1580 916340 N/A N/A  5650  5665TGGATATATTGGGCTC 97 1581 916360 N/A N/A  6603  6618 ACATTCAGGGTTTACA 181582 916380 N/A N/A  7468  7483 GTATTCATTAGCATCA 51 1583 916400 N/A N/A 7688  7703 AGGTTAATCCATGGGT 29 1584 916560 N/A N/A 12287 12302GAGTTTTAGGTCTGGG 95 1585 916580 N/A N/A 12905 12920 CTTATAAAGCACACGG 951586 916600 N/A N/A 13683 13698 GGGCATGGCTGATCCT  8 1587 916620 N/A N/A14099 14114 CAAACTTGTCTAGTGG 67 1588 916640 N/A N/A 14600 14615TCGCATCCATGGGTCC 83 1589 916660 N/A N/A 15429 15444 GCTCTTACAGCATTGG 511590 916680 N/A N/A 15974 15989 CCATCTACCAAACTGC 61 1591 916700 N/A N/A17195 17210 GACTTAGTCCGTGTTC 49 1592 916720 N/A N/A 17883 17898CAGCATCTATGTTCTC 67 1593 916740 N/A N/A 19239 19254 ATAGACTGTGAGCTGT 821594 916759 N/A N/A 20354 20369 GACCATTCTGCTCCCC 20 1595 916779 N/A N/A20632 20647 GCCCATACCTTTTATC 47 1596 916799 N/A N/A 20987 21002GTATTAAACTGCCAAG 81 1597 916819 N/A N/A 21444 21459 CTAACCAATATACTGA 731598 916839 N/A N/A 22506 22521 GGCTGGTGATGAAACA  0 1599 916859 N/A N/A23345 23360 CCTCATGGTTTGCTGT 31 1600 916879 N/A N/A 24273 24288CAGTACAACAGAGGGT 81 1601 916899 N/A N/A 25064 25079 CACATTGCCGGCCAGT 581602

TABLE 24Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915362   44   59  2782  2797CTCAGTGTCTCGGCCA  0 1603 915382  106  121  2844  2859 ATCGGCTCGGGTCCTG30 1604 915402  189  204  2927  2942 ACAAGCTCCAGCCGCG 24 1605 915422 248  263  2986  3001 GCTCAGGCAGCGGGTC 33 1606 915442  346  361 N/A N/ATCCAGCGGGATACCGG  0 1607 915462  385  400  5959  5974 GCCTTCCGCACAAGAT60 1608 915482  415  430  5989  6004 GGATGGAAGATGCCAA 60 1609 915502 451  466  6025  6040 AGACCCTGTCGGAGGA 75 1610 915522  487  502  6061 6076 ATGAGCTGGTGGACAT 74 1611 915542  509  524  6083  6098AGAGATGCCTATTTTG 91 1612 915562  558  573  6132  6147 ACCGAAAGTCAGACAC 0 1613 915602  692  707 11910 11925 GGCATCAATGAAGGGT 88 1614 915622 752  767 11970 11985 CTTGACTTTAGGGCAG 86 1615 915642  824  839 1204212057 GAGAAGGTAGAGGTTC 81 1616 915662  878  893 13614 13629TCGAAGGCATATCTCT 16 1617 915682  931  946 16051 16066 GGCCTGTTGCAGATGC 0 1618 915702  984  999 16104 16119 GCATGGCGACCTCAGG 24 1619 9157221036 1051 16156 16171 GCCAAGGCAGCCGACT  0 1620 915742 1123 1138 1624316258 GGCGAGAGGGTGTCCA  0 1621 915762 1173 1188 19013 19028TGTATCCACCTTTGTC 85 1622 915782 1228 1243 19068 19083 GGCAGCATTACATAAG73 1623 915802 1283 1298 N/A N/A CCATGTCACCAGTCTC 59 1624 915822 13471362 23752 23767 CTCGAGTGAACACCTG  0 1625 915842 1404 1419 25167 25182CCTGTTGGCTGCTCAC 88 1626 915862 1472 1487 25235 25250 TGCTGGACAGCCCTTG 0 1627 915882 1541 1556 25304 25319 TTTATTGCCCAAGAAG 38 1628 9159021600 1615 25363 25378 AGACTCTTCTCTAGTG 60 1629 915922 1632 1647 2539525410 ATCTGCTAGACTCGCC 86 1630 915942 1684 1699 25447 25462GCAATGCGGAGGTAGC 38 1631 915962 1767 1782 25530 25545 AAAGGTTGCTTCCTAG77 1632 915982 1787 1802 25550 25565 GCTGGACCGCTGCACA 79 1633 9160021814 1829 25577 25592 ACGCATGCTGATGTAT 72 1634 916022 1841 1856 2560425619 GGTGTCATTTCCCAAC 80 1635 916042 1883 1898 25646 25661CCACGAAACAGTCAGT 87 1636 916062 1912 1927 25675 25690 ATGCTGGAACAGTCTG78 1637 916082 1943 1958 25706 25721 CCATCCAAACACCTGT 64 1638 9161021976 1991 25739 25754 GGGCCAGCCTACCCCC  0 1639 916122 2025 2040 2578825803 GAAGTGGGATCATGCT 71 1640 916141 2097 2112 25860 25875ATCATCTTTGCAGACC 91 1641 916161 2138 2153 25901 25916 TTGTTACCCCCGCCAT89 1642 916181 2259 2274 26022 26037 CTGATTCACATAATAC 91 1643 9162012623 2638 26386 26401 CCTGCACACTAGATTA 59 1644 916221 2676 2691 2643926454 AGGCGGAAGCTCCTGC  0 1645 916241 2706 2721 26469 26484AGGTTCAAGTTGTGTG 87 1646 916281 N/A N/A  4224  4239 AATGTACGGAATCTCA 831647 916301 N/A N/A  4810  4825 GTCCATGTGGGTGTCC 74 1648 916321 N/A N/A 5399  5414 GGCCTTAATGCTATCA 13 1649 916341 N/A N/A  5711  5726TAGTATGAAATATCTC 96 1650 916361 N/A N/A  6862  6877 ATTGTAACTGCCAGGC  01651 916381 N/A N/A  7471  7486 CCGGTATTCATTAGCA  0 1652 916401 N/A N/A 7728  7743 GAGCAGGGCAACAAAC 22 1653 916561 N/A N/A 12315 12330ATATAACCACAGCCTG 54 1654 916581 N/A N/A 12906 12921 GCTTATAAAGCACACG 941655 916601 N/A N/A 13702 13717 TAGTAAATGCTTGTCA 95 1656 916621 N/A N/A14123 14138 GGCAGAAATGTGCTCT 60 1657 916641 N/A N/A 14632 14647CTTCATGCCATCCTGT 83 1658 916661 N/A N/A 15430 15445 TGCTCTTACAGCATTG  01659 916681 N/A N/A 16262 16277 GGTACCTGTAGCGAGC  0 1660 916701 N/A N/A17197 17212 TTGACTTAGTCCGTGT 93 1661 916721 N/A N/A 18220 18235AGCTACATCAGGCTGG  0 1662 916741 N/A N/A 19244 19259 TGCACATAGACTGTGA  01663 916760 N/A N/A 20373 20388 GACTGCTGAGCCAAGC 61 1664 916780 N/A N/A20657 20672 AGAAATTGCAGTGCCC 92 1665 916800 N/A N/A 20988 21003GGTATTAAACTGCCAA  0 1666 916820 N/A N/A 21447 21462 TCACTAACCAATATAC 471667 916840 N/A N/A 22602 22617 ATAAATCTGCAAGAGC 62 1668 916860 N/A N/A23369 23384 TCTCATGGTCAAGACC 52 1669 916880 N/A N/A 24305 24320GACTGCTAGGCTTCAC 54 1670 916900 N/A N/A 25100 25115 CGCTGCTGCAGTGTGC 341671

Human primer probe set RTS36075 (forward sequence TGAGGCTGGAGGGAGATG,designated herein as SEQ ID NO: 14; reverse sequenceGCTCATGTATCCACCTTTGTCT, designated herein as SEQ ID NO: 15; probesequence CTAGACCACCTGCGTCTCAGCATC, designated herein as SEQ ID NO: 16)was also used to measure mRNA levels. PNPLA3 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of PNPLA3, relative to untreated controlcells.

TABLE 25Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 898558  581  596 N/A N/AGGCATCCACGACTTCG 87 1672 912709   27   42  2765  2780 GGCATTCCCAGCGCGA 0   17 912710   95  110  2833  2848 TCCTGATCCGCAGCAG  0   18 912711 103  118  2841  2856 GGCTCGGGTCCTGATC  0   19 912712  131  146  2869 2884 GTTAGGATCTGGGTCG 76   20 912713  164  179  2902  2917GTACATGGCGGCGGCG  0   21 912714  183  198  2921  2936 TCCAGCCGCGCTCTGC29   22 912715  196  211  2934  2949 GCGAAGGACAAGCTCC 31   23 912716 197  212  2935  2950 CGCGAAGGACAAGCTC  0   24 912717  272  287  3010 3025 GCGGAGGAGGTGCGGG  0   25 912718  273  288  3011  3026CGCGGAGGAGGTGCGG  0   26 912719  274  289  3012  3027 TCGCGGAGGAGGTGCG16   27 912720  290  305  3028  3043 GAACAACATGCGCGCG  0   28 912721 291  306  3029  3044 CGAACAACATGCGCGC  2   29 912722  292  307  3030 3045 CCGAACAACATGCGCG  0   30 912723  293  308  3031  3046GCCGAACAACATGCGC  0   31 912724  294  309  3032  3047 CGCCGAACAACATGCG 0   32 912725  323  338  3061  3076 GCCGACGCAGTGCAAC  0   33 912726 324  339  3062  3077 CGCCGACGCAGTGCAA  0   34 912727  340  355  3078 3093 GGGATACCGGAGAGGA 32   35 912728  370  385  5944  5959TCTGAGAGGACCTGCA 31   36 912729  375  390  5949  5964 CAAGATCTGAGAGGAC60   37 912730  404  419  5978  5993 GCCAATGTTCCGACTC 52   38 912731 410  425  5984  5999 GAAGATGCCAATGTTC 31   39 912732  429  444  6003 6018 TTAAGTTGAAGGATGG 93   40 912733  432  447  6006  6021TGCTTAAGTTGAAGGA 82   41 912734  478  493  6052  6067 TGGACATTGGCCGGGA73   42 912735  479  494  6053  6068 GTGGACATTGGCCGGG 44   43 912736 484  499  6058  6073 AGCTGGTGGACATTGG 29   44 912737  528  543  6102 6117 CATCAGACACTCTGGT  0   45 912738  531  546  6105  6120CCCCATCAGACACTCT 55   46 912739  552  567  6126  6141 AGTCAGACACCAGAAC23   47 912740  582  597 N/A N/A AGGCATCCACGACTTC 40 1673 912741  584 599 N/A N/A CAAGGCATCCACGACT 55 1674 912742  591  606 N/A N/AAACATACCAAGGCATC 59 1675 912743  593  608 N/A N/A GGAACATACCAAGGCA 691676 912744  594  609  7824  7839 AGGAACATACCAAGGC 85 1677 912745  625 640  7855  7870 GGGATAAGGCCACTGT 71 1678 912746  626  641  7856  7871AGGGATAAGGCCACTG 12 1679 912747  630  645  7860  7875 AAGGAGGGATAAGGCC 0 1680 912748  652  667 N/A N/A ACATATCGCACGCCTC 35 1681 912749  653 668 N/A N/A CACATATCGCACGCCT  3 1682 912750  654  669 N/A N/ACCACATATCGCACGCC 27 1683 912751  656  671 N/A N/A ATCCACATATCGCACG 241684 912752  660  675 11878 11893 CTCCATCCACATATCG 87 1685 912753  689 704 11907 11922 ATCAATGAAGGGTACG 79 1686 912754  690  705 11908 11923CATCAATGAAGGGTAC 63 1687 912755  693  708 11911 11926 TGGCATCAATGAAGGG68   48 912756  698  713 11916 11931 TGTTTTGGCATCAATG 88   49 912757 746  761 11964 11979 TTTAGGGCAGATGTCG 75   50 912758  747  762 1196511980 CTTTAGGGCAGATGTC 82   51 912759  795  810 12013 12028GTAGACTGAGCTTGGT 96   52 912760  820  835 12038 12053 AGGTAGAGGTTCCCTG 0   53 912761  841  856 12059 12074 GGGACAAAAGCTCTCG  0   54 912762 873  888 13609 13624 GGCATATCTCTCCCAG  0   55 912763  874  889 1361013625 AGGCATATCTCTCCCA  0   56 912764  886  901 13622 13637AAATATCCTCGAAGGC 71   57 912765  888  903 13624 13639 CCAAATATCCTCGAAG37   58 912766  889  904 13625 13640 TCCAAATATCCTCGAA  0   59 912767 894  909 13630 13645 ATGCATCCAAATATCC 42   60 912768  925  940 N/A N/ATTGCAGATGCCCTTCT  5   61 912769  968  983 16088 16103 ATCCATCCCTTCTGAG 6   62 912770  986 1001 16106 16121 GGGCATGGCGACCTCA  0   63 9127711004 1019 16124 16139 ACTCATGTTTGCCCAG 67   64 912782 1195 1210 1903519050 AGCAAGTTGCAAATCT 71   75 912783 1199 1214 19039 19054GGGTAGCAAGTTGCAA 37   76 912784 1205 1220 19045 19060 CCTAATGGGTAGCAAG25   77 912785 1206 1221 19046 19061 TCCTAATGGGTAGCAA 64   78

TABLE 26Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 912786 1207 1222 19047 19062ATCCTAATGGGTAGCA 65  79 912787 1211 1226 19051 19066 CATTATCCTAATGGGT 43 80 912788 1212 1227 19052 19067 ACATTATCCTAATGGG  0  81 912789 12131228 19053 19068 GACATTATCCTAATGG 59  82 912790 1220 1235 19060 19075TACATAAGACATTATC  8  83 912791 1224 1239 19064 19079 GCATTACATAAGACAT 86 84 912792 1245 1260 19085 19100 CCACAGGCAGGGTACA 58  85 912793 12461261 19086 19101 TCCACAGGCAGGGTAC  5  86 912794 1253 1268 19093 19108GGCAGATTCCACAGGC 68  87 912795 1259 1274 19099 19114 CGCAATGGCAGATTCC 84 88 912796 1265 1280 19105 19120 GACAATCGCAATGGCA 63  89 912797 12661281 19106 19121 GGACAATCGCAATGGC 54  90 912798 1267 1282 19107 19122TGGACAATCGCAATGG 59  91 912799 1285 1300 23690 23705 AGCCATGTCACCAGTC 51 92 912800 1289 1304 23694 23709 TGGAAGCCATGTCACC 32  93 912801 12901305 23695 23710 CTGGAAGCCATGTCAC 44  94 912802 1297 1312 23702 23717GGCATATCTGGAAGCC  0  95 912803 1298 1313 23703 23718 GGGCATATCTGGAAGC  0 96 912804 1351 1366 23756 23771 AGCACTCGAGTGAACA  6  97 912805 13861401 N/A N/A GCATTTGGGACCTGGA 54  98 912806 1387 1402 N/A N/AGGCATTTGGGACCTGG 33  99 912807 1388 1403 25151 25166 TGGCATTTGGGACCTG  0100 912808 1394 1409 25157 25172 GCTCACTGGCATTTGG  7 101 912809 15231538 25286 25301 GTTCAGGCTGGACCTG 17 102 912810 1547 1562 25310 25325AGGTACTTTATTGCCC 30 103 912811 1550 1565 25313 25328 AGCAGGTACTTTATTG 55104 912812 1653 1668 25416 25431 AACTTTAGCACCTCTG 87 105 912813 16551670 25418 25433 GAAACTTTAGCACCTC 85 106 912814 1656 1671 25419 25434GGAAACTTTAGCACCT 26 107 912815 1669 1684 25432 25447 CTGCACAAAGATGGGA 66108 912816 1671 1686 25434 25449 AGCTGCACAAAGATGG 41 109 912817 16851700 25448 25463 AGCAATGCGGAGGTAG 35 110 912818 1740 1755 25503 25518ACCAACTCAGCTCAGA 76 111 912819 1741 1756 25504 25519 AACCAACTCAGCTCAG 77112 912820 1757 1772 25520 25535 TCCTAGCTTTTCATAA 18 113 912821 17881803 25551 25566 TGCTGGACCGCTGCAC  1 114 912822 1796 1811 25559 25574GAGTTAAGTGCTGGAC 90 115 912823 1802 1817 25565 25580 GTATTAGAGTTAAGTG 86116 912824 1803 1818 25566 25581 TGTATTAGAGTTAAGT 79 117 912825 18061821 25569 25584 TGATGTATTAGAGTTA 89 118 912826 1808 1823 25571 25586GCTGATGTATTAGAGT 79 119 912827 1821 1836 25584 25599 TGAATTAACGCATGCT 73120 912828 1822 1837 25585 25600 CTGAATTAACGCATGC 69 121 912829 18701885 25633 25648 AGTAAGGGACCCTCTG  0 122 912830 1871 1886 25634 25649CAGTAAGGGACCCTCT 44 123 912831 1872 1887 25635 25650 TCAGTAAGGGACCCTC 67124 912832 1874 1889 25637 25652 AGTCAGTAAGGGACCC 50 125 912833 18931908 25656 25671 ATTAATAGGGCCACGA 78 126 912834 1895 1910 25658 25673CCATTAATAGGGCCAC 72 127 912835 1896 1911 25659 25674 ACCATTAATAGGGCCA 65128 912836 1906 1921 25669 25684 GAACAGTCTGACCATT 82 129 912837 19081923 25671 25686 TGGAACAGTCTGACCA 39 130 912838 1909 1924 25672 25687CTGGAACAGTCTGACC 84 131 912839 1911 1926 25674 25689 TGCTGGAACAGTCTGA 72132 912840 1916 1931 25679 25694 CCTCATGCTGGAACAG 84 133 912841 19281943 25691 25706 TCATTCTAAGAACCTC 87 134 912842 1945 1960 25708 25723ACCCATCCAAACACCT 18 135 912843 1982 1997 25745 25760 ACACATGGGCCAGCCT 46136 912844 1989 2004 25752 25767 CAAGATCACACATGGG 71 137 912845 20572072 25820 25835 GGGACGAACTGCACCC  0 138 912846 2098 2113 25861 25876TATCATCTTTGCAGAC 68 139 912847 2116 2131 25879 25894 GTTTTTAGTAGTCAAG 90140 912848 2117 2132 25880 25895 CGTTTTTAGTAGTCAA 94 141 912849 21452160 25908 25923 TATCATCTTGTTACCC 87 142 912850 2148 2163 25911 25926GATTATCATCTTGTTA 60 143 912851 2150 2165 25913 25928 TAGATTATCATCTTGT 50144 912852 2151 2166 25914 25929 GTAGATTATCATCTTG 72 145 912853 21522167 25915 25930 AGTAGATTATCATCTT 79 146 912854 2175 2190 25938 25953GTGAAAAAGGTGTTCT 64 147 912855 2182 2197 25945 25960 TAGTTAGGTGAAAAAG 77148 912856 2188 2203 25951 25966 TTATTTTAGTTAGGTG 82 149 912857 21902205 25953 25968 CATTATTTTAGTTAGG 77 150 912858 2273 2288 26036 26051CTACTAACATCTCACT 48 151 912859 2274 2289 26037 26052 TCTACTAACATCTCAC 91152 912860 2278 2293 26041 26056 TTATTCTACTAACATC 37 153 912861 22802295 26043 26058 GCTTATTCTACTAACA 77 154 912862 2281 2296 26044 26059GGCTTATTCTACTAAC 70 155 912863 2632 2647 26395 26410 GGTGAATGCCCTGCAC 42156

Study 2

Cultured A431 cells at a density of 5,000 cells per well weretransfected by free uptake with 1,000 nM antisense oligonucleotide.After a treatment period of approximately 24 hours, RNA was isolatedfrom the cells and PNPLA3 mRNA levels were measured by quantitativereal-time PCR. Human primer probe set RTS36070 was used to measure mRNAlevels. PNPLA3 mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN®. Results are presented as percent inhibitionof PNPLA3, relative to untreated control cells.

TABLE 27Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 97  702 959270  413  428  5987  6002 ATGGAAGATGCCAATG32 1688 959280  491  506  6065  6080 GGAGATGAGCTGGTGG 66 1689 959290 793  808 12011 12026 AGACTGAGCTTGGTGA 78 1690 959300  899  914 1363513650 CCTGAATGCATCCAAA 69 1691 959310 1084 1099 16204 16219ATGCTGAGACGCAGGT  0 1692 959320 1256 1271 19096 19111 AATGGCAGATTCCACA25 1693 959330 1642 1657 25405 25420 CTCTGAAAGAATCTGC 75 1694 9593401659 1674 25422 25437 ATGGGAAACTTTAGCA 77 1695 959350 1839 1854 2560225617 TGTCATTTCCCAACCA 79 1696 959360 2114 2129 25877 25892TTTTAGTAGTCAAGGT 88 1697 959370 2223 2238 25986 26001 GCTTCCTTACATTTTT85 1698 959380 2269 2284 26032 26047 TAACATCTCACTGATT 42 1699 959390 N/AN/A  4311  4326 CTAGTGAGAAACAAAC  0 1700 959400 N/A N/A  4761  4776TTATTGTTGCTAAACC 32 1701 959410 N/A N/A  4863  4878 ACTTTAGGCTCCTGGG 601702 959420 N/A N/A  5285  5300 AGCCATAAATCTTGGG 24 1703 959430 N/A N/A 5573  5588 ATGACATCATGGCTTC 93 1704 959440 N/A N/A  5603  5618TTATTCAATGTGGCTT 95 1705 959450 N/A N/A  5640  5655 GGGCTCAATGAAATTA 121706 959460 N/A N/A  5713  5728 CTTAGTATGAAATATC 86 1707 959470 N/A N/A 5808  5823 TACTGTCTACTATGGG 91 1708 959480 N/A N/A  6157  6172CTTACATCCACGACTT 35 1709 959660 N/A N/A 12153 12168 CAGTAACTGGTAGCTC 741710 959670 N/A N/A 12169 12184 TGTTTGATTGTGCAGA 95 1711 959680 N/A N/A12210 12225 CGCCTTTTATTTCCGT 92 1712 959690 N/A N/A 12313 12328ATAACCACAGCCTGGG 66 1713 959700 N/A N/A 12675 12690 ATAAGAATCATCTTAG  71714 959710 N/A N/A 12711 12726 ACTACCGAACGCAGTT 41 1715 959720 N/A N/A12757 12772 TAGAGTGGTAAGGCAT 84 1716 959730 N/A N/A 12793 12808GGTTGGTGTACATGGT 96 1717 959740 N/A N/A 12880 12895 TCCTGTTAGACAGCTT 931718 959750 N/A N/A 12902 12917 ATAAAGCACACGGGAA 86 1719 959760 N/A N/A12931 12946 TAAGAGCTGTCTCCTC 85 1720 959770 N/A N/A 12972 12987CTAACAAACTTTGCAG 79 1721 959780 N/A N/A 13392 13407 TGTCACCCTTCCACGG 151722 959790 N/A N/A 13526 13541 ATTGGAAGACCGCAGA 43 1723 959800 N/A N/A13706 13721 CCGCTAGTAAATGCTT 44 1724 959810 N/A N/A 13737 13752AACTAAGGCAAATCTC 77 1725 959820 N/A N/A 13915 13930 GAGTCATGACATCCCA 891726 959830 N/A N/A 14299 14314 GCAGATAAATACACAT 93 1727 959840 N/A N/A14424 14439 TTTCCCATCGACACAG 78 1728 959850 N/A N/A 14571 14586GCTCCTACTTATCCCC 76 1729 959860 N/A N/A 15202 15217 TATTGCCAGGTATCTG 641730 959870 N/A N/A 15599 15614 CAATACATAGCAGAGC 23 1731 959880 N/A N/A17192 17207 TTAGTCCGTGTTCAGG 90 1732 959890 N/A N/A 17222 17237GTAGCTGGTTTGTGGG 20 1733 959900 N/A N/A 17295 17310 CATCTCTTAGGGCACC 791734 959910 N/A N/A 18393 18408 GTTTGGAAGTCGCCAT 77 1735 959920 N/A N/A20284 20299 AGGAAGCCCAATCAAG 85 1736 959930 N/A N/A 20512 20527CAGATTGAGTCTCCTG 10 1737 959940 N/A N/A 20607 20622 TTTTACGATCATCATT 721738 959950 N/A N/A 20661 20676 GCTTAGAAATTGCAGT 75 1739 959960 N/A N/A20812 20827 AGGGTAATATTCAGAC 86 1740 959970 N/A N/A 20934 20949TGTAGCAGACAGATCA 74 1741 959980 N/A N/A 21000 21015 TTTAACAGCTCAGGTA 661742 959990 N/A N/A 21405 21420 ATTCTAGACATGGCCA 51 1743 960000 N/A N/A21442 21457 AACCAATATACTGAGA 71 1744 960010 N/A N/A 21545 21560AGACATATGACATTTC 91 1745 960020 N/A N/A 22765 22780 ACATGACAGACTAACT 551746 960030 N/A N/A 24039 24054 CATCAATGCTGCACTC 13 1747

TABLE 28Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959271  425  440  5999  6014 GTTGAAGGATGGATGG90 1748 959281  511  526  6085  6100 AGAGAGATGCCTATTT 73 1749 959291 813  828 12031 12046 GGTTCCCTGTGCAGAG 79 1750 959301  904  919 1364013655 AAGAACCTGAATGCAT 48 1751 959311 1085 1100 16205 16220GATGCTGAGACGCAGG  0 1752 959321 1602 1617 25365 25380 ACAGACTCTTCTCTAG45 1753 959331 1643 1658 25406 25421 CCTCTGAAAGAATCTG 81 1754 9593411673 1688 25436 25451 GTAGCTGCACAAAGAT 69 1755 959351 1842 1857 2560525620 TGGTGTCATTTCCCAA 19 1756 959361 2115 2130 25878 25893TTTTTAGTAGTCAAGG 91 1757 959371 2240 2255 26003 26018 ATTCAACAGGTAACAA69 1758 959381 2271 2286 26034 26049 ACTAACATCTCACTGA 33 1759 959391 N/AN/A  4313  4328 AGCTAGTGAGAAACAA 47 1760 959401 N/A N/A  4764  4779CTTTTATTGTTGCTAA 77 1761 959411 N/A N/A  4868  4883 AGTGTACTTTAGGCTC 901762 959421 N/A N/A  5286  5301 CAGCCATAAATCTTGG  0 1763 959431 N/A N/A 5574  5589 AATGACATCATGGCTT 74 1764 959441 N/A N/A  5604  5619TTTATTCAATGTGGCT 96 1765 959451 N/A N/A  5642  5657 TTGGGCTCAATGAAAT  01766 959461 N/A N/A  5714  5729 GCTTAGTATGAAATAT 78 1767 959471 N/A N/A 5809  5824 GTACTGTCTACTATGG 78 1768 959481 N/A N/A  6160  6175CTGCTTACATCCACGA  4 1769 959661 N/A N/A 12154 12169 ACAGTAACTGGTAGCT 721770 959671 N/A N/A 12170 12185 CTGTTTGATTGTGCAG 50 1771 959681 N/A N/A12211 12226 GCGCCTTTTATTTCCG 14 1772 959691 N/A N/A 12322 12337CCTGACTATATAACCA 56 1773 959701 N/A N/A 12689 12704 GACCGTGTTTCCAAAT 971774 959711 N/A N/A 12712 12727 AACTACCGAACGCAGT 48 1775 959721 N/A N/A12759 12774 GGTAGAGTGGTAAGGC 95 1776 959731 N/A N/A 12828 12843CATAGCCTTCTTTCTT 93 1777 959741 N/A N/A 12882 12897 AATCCTGTTAGACAGC 901778 959751 N/A N/A 12903 12918 TATAAAGCACACGGGA 82 1779 959761 N/A N/A12933 12948 AATAAGAGCTGTCTCC 87 1780 959771 N/A N/A 12974 12989CCCTAACAAACTTTGC 62 1781 959781 N/A N/A 13394 13409 AATGTCACCCTTCCAC 901782 959791 N/A N/A 13527 13542 CATTGGAAGACCGCAG 76 1783 959801 N/A N/A13707 13722 ACCGCTAGTAAATGCT 35 1784 959811 N/A N/A 13740 13755TAGAACTAAGGCAAAT 65 1785 959821 N/A N/A 13916 13931 GGAGTCATGACATCCC 421786 959831 N/A N/A 14302 14317 TGAGCAGATAAATACA 75 1787 959841 N/A N/A14425 14440 CTTTCCCATCGACACA 79 1788 959851 N/A N/A 14573 14588GCGCTCCTACTTATCC  0 1789 959861 N/A N/A 15203 15218 ATATTGCCAGGTATCT 671790 959871 N/A N/A 15763 15778 GTGTTGGTTTATAACA 13 1791 959881 N/A N/A17194 17209 ACTTAGTCCGTGTTCA 45 1792 959891 N/A N/A 17224 17239CTGTAGCTGGTTTGTG 42 1793 959901 N/A N/A 17296 17311 CCATCTCTTAGGGCAC 531794 959911 N/A N/A 18394 18409 TGTTTGGAAGTCGCCA 87 1795 959921 N/A N/A20289 20304 ATCAGAGGAAGCCCAA 84 1796 959931 N/A N/A 20514 20529ACCAGATTGAGTCTCC 91 1797 959941 N/A N/A 20613 20628 CTCTAATTTTACGATC 701798 959951 N/A N/A 20662 20677 AGCTTAGAAATTGCAG  0 1799 959961 N/A N/A20813 20828 CAGGGTAATATTCAGA 87 1800 959971 N/A N/A 20936 20951TTTGTAGCAGACAGAT 18 1801 959981 N/A N/A 21001 21016 TTTTAACAGCTCAGGT 711802 959991 N/A N/A 21406 21421 AATTCTAGACATGGCC 25 1803 960001 N/A N/A21443 21458 TAACCAATATACTGAG 72 1804 960011 N/A N/A 22023 22038CGCAAAAAGACAACGA 16 1805 960021 N/A N/A 22766 22781 GACATGACAGACTAAC 761806 960031 N/A N/A 24040 24055 CCATCAATGCTGCACT 61 1807

TABLE 29Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 97  702 959272  426  441  6000  6015 AGTTGAAGGATGGATG76 1808 959282  517  532  6091  6106 CTGGTAAGAGAGATGC 64 1809 959292 815  830 12033 12048 GAGGTTCCCTGTGCAG 81 1810 959302  905  920 1364113656 CAAGAACCTGAATGCA 65 1811 959312 1087 1102 16207 16222AGGATGCTGAGACGCA 66 1812 959322 1604 1619 25367 25382 TCACAGACTCTTCTCT50 1813 959332 1644 1659 25407 25422 ACCTCTGAAAGAATCT 69 1814 9593421675 1690 25438 25453 AGGTAGCTGCACAAAG 78 1815 959352 1903 1918 2566625681 CAGTCTGACCATTAAT 77 1816 959362 2173 2188 25936 25951GAAAAAGGTGTTCTAA 85 1817 959372 2242 2257 26005 26020 AAATTCAACAGGTAAC62 1818 959382 2275 2290 26038 26053 TTCTACTAACATCTCA 80 1819 959392 N/AN/A  4732  4747 ATGATCATGTGGCGGT 80 1820 959402 N/A N/A  4765  4780ACTTTTATTGTTGCTA 86 1821 959412 N/A N/A  4869  4884 GAGTGTACTTTAGGCT 941822 959422 N/A N/A  5389  5404 CTATCAGGTGCAGGAG 93 1823 959432 N/A N/A 5575  5590 CAATGACATCATGGCT 90 1824 959442 N/A N/A  5607  5622TACTTTATTCAATGTG  0 1825 959452 N/A N/A  5643  5658 ATTGGGCTCAATGAAA 351826 959462 N/A N/A  5716  5731 TGGCTTAGTATGAAAT 80 1827 959472 N/A N/A 5864  5879 TTTGGCAAGGCCAGAA  0 1828 959482 N/A N/A  6960  6975GCATAGAGGAAGCTCG 32 1829 959662 N/A N/A 12155 12170 GACAGTAACTGGTAGC 921830 959672 N/A N/A 12172 12187 TTCTGTTTGATTGTGC 97 1831 959682 N/A N/A12280 12295 AGGTCTGGGTATATGT 93 1832 959692 N/A N/A 12323 12338CCCTGACTATATAACC 32 1833 959702 N/A N/A 12691 12706 TTGACCGTGTTTCCAA 941834 959712 N/A N/A 12713 12728 AAACTACCGAACGCAG 92 1835 959722 N/A N/A12760 12775 TGGTAGAGTGGTAAGG 94 1836 959732 N/A N/A 12829 12844GCATAGCCTTCTTTCT 87 1837 959742 N/A N/A 12883 12898 CAATCCTGTTAGACAG 131838 959752 N/A N/A 12904 12919 TTATAAAGCACACGGG 87 1839 959762 N/A N/A12934 12949 CAATAAGAGCTGTCTC 81 1840 959772 N/A N/A 13370 13385TGCAGGCACCCCAGCA  0 1841 959782 N/A N/A 13395 13410 GAATGTCACCCTTCCA 901842 959792 N/A N/A 13528 13543 TCATTGGAAGACCGCA 84 1843 959802 N/A N/A13708 13723 GACCGCTAGTAAATGC 57 1844 959812 N/A N/A 13741 13756TTAGAACTAAGGCAAA 78 1845 959822 N/A N/A 13917 13932 TGGAGTCATGACATCC  01846 959832 N/A N/A 14303 14318 CTGAGCAGATAAATAC 74 1847 959842 N/A N/A14553 14568 AGTCTTAATGTGGATT 76 1848 959852 N/A N/A 14667 14682AGTGTCCCCATCCCCA 54 1849 959862 N/A N/A 15204 15219 AATATTGCCAGGTATC 561850 959872 N/A N/A 15765 15780 TAGTGTTGGTTTATAA 89 1851 959882 N/A N/A17196 17211 TGACTTAGTCCGTGTT 43 1852 959892 N/A N/A 17225 17240TCTGTAGCTGGTTTGT 61 1853 959902 N/A N/A 17298 17313 TCCCATCTCTTAGGGC 241854 959912 N/A N/A 18396 18411 TATGTTTGGAAGTCGC 91 1855 959922 N/A N/A20290 20305 AATCAGAGGAAGCCCA 40 1856 959932 N/A N/A 20515 20530AACCAGATTGAGTCTC 72 1857 959942 N/A N/A 20614 20629 TCTCTAATTTTACGAT 231858 959952 N/A N/A 20663 20678 CAGCTTAGAAATTGCA 24 1859 959962 N/A N/A20814 20829 CCAGGGTAATATTCAG 87 1860 959972 N/A N/A 20937 20952CTTTGTAGCAGACAGA 50 1861 959982 N/A N/A 21003 21018 TATTTTAACAGCTCAG 941862 959992 N/A N/A 21409 21424 TGCAATTCTAGACATG 12 1863 960002 N/A N/A21445 21460 ACTAACCAATATACTG 55 1864 960012 N/A N/A 22541 22556CAACAGATTACTGGAC 28 1865 960022 N/A N/A 22768 22783 AGGACATGACAGACTA 661866 960032 N/A N/A 24041 24056 ACCATCAATGCTGCAC 79 1867

TABLE 30Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959273  427  442  6001  6016 AAGTTGAAGGATGGAT86 1868 959283  694  709 11912 11927 TTGGCATCAATGAAGG 73 1869 959293 816  831 12034 12049 AGAGGTTCCCTGTGCA 80 1870 959303  906  921 1364213657 CCAAGAACCTGAATGC 65 1871 959313 1090 1105 16210 16225GGCAGGATGCTGAGAC 43 1872 959323 1605 1620 25368 25383 CTCACAGACTCTTCTC81 1873 959333 1646 1661 25409 25424 GCACCTCTGAAAGAAT 51 1874 9593431677 1692 25440 25455 GGAGGTAGCTGCACAA 73 1875 959353 1904 1919 2566725682 ACAGTCTGACCATTAA 85 1876 959363 2179 2194 25942 25957TTAGGTGAAAAAGGTG 91 1877 959373 2258 2273 26021 26036 TGATTCACATAATACA71 1878 959383 2277 2292 26040 26055 TATTCTACTAACATCT 38 1879 959393 N/AN/A  4733  4748 AATGATCATGTGGCGG 93 1880 959403 N/A N/A  4767  4782TGACTTTTATTGTTGC 87 1881 959413 N/A N/A  4870  4885 TGAGTGTACTTTAGGC 941882 959423 N/A N/A  5392  5407 ATGCTATCAGGTGCAG  0 1883 959433 N/A N/A 5578  5593 GCACAATGACATCATG 86 1884 959443 N/A N/A  5608  5623TTACTTTATTCAATGT  9 1885 959453 N/A N/A  5644  5659 TATTGGGCTCAATGAA 801886 959463 N/A N/A  5798  5813 TATGGGAGCCACATGT  4 1887 959473 N/A N/A 5866  5881 CTTTTGGCAAGGCCAG  0 1888 959483 N/A N/A  7199  7214TTAAACAGAGGATGCA 31 1889 959663 N/A N/A 12156 12171 AGACAGTAACTGGTAG 751890 959673 N/A N/A 12199 12214 TCCGTTAACCATCAAG 95 1891 959683 N/A N/A12282 12297 TTAGGTCTGGGTATAT 94 1892 959693 N/A N/A 12324 12339CCCCTGACTATATAAC  0 1893 959703 N/A N/A 12692 12707 CTTGACCGTGTTTCCA 971894 959713 N/A N/A 12715 12730 TTAAACTACCGAACGC 95 1895 959723 N/A N/A12761 12776 ATGGTAGAGTGGTAAG 77 1896 959733 N/A N/A 12832 12847ATTGCATAGCCTTCTT 95 1897 959743 N/A N/A 12884 12899 CCAATCCTGTTAGACA 831898 959753 N/A N/A 12908 12923 CTGCTTATAAAGCACA  2 1899 959763 N/A N/A12935 12950 ACAATAAGAGCTGTCT 85 1900 959773 N/A N/A 13372 13387TTTGCAGGCACCCCAG 63 1901 959783 N/A N/A 13396 13411 TGAATGTCACCCTTCC 531902 959793 N/A N/A 13531 13546 GCATCATTGGAAGACC 86 1903 959803 N/A N/A13713 13728 ACCAAGACCGCTAGTA 38 1904 959813 N/A N/A 13743 13758TGTTAGAACTAAGGCA 79 1905 959823 N/A N/A 13919 13934 CCTGGAGTCATGACAT  71906 959833 N/A N/A 14304 14319 TCTGAGCAGATAAATA 39 1907 959843 N/A N/A14554 14569 AAGTCTTAATGTGGAT 84 1908 959853 N/A N/A 14669 14684TTAGTGTCCCCATCCC 78 1909 959863 N/A N/A 15205 15220 GAATATTGCCAGGTAT 861910 959873 N/A N/A 15766 15781 TTAGTGTTGGTTTATA 92 1911 959883 N/A N/A17198 17213 TTTGACTTAGTCCGTG 86 1912 959893 N/A N/A 17226 17241CTCTGTAGCTGGTTTG 82 1913 959903 N/A N/A 17601 17616 TTGATAGTGAATGTGT 831914 959913 N/A N/A 18397 18412 ATATGTTTGGAAGTCG 91 1915 959923 N/A N/A20291 20306 CAATCAGAGGAAGCCC 34 1916 959933 N/A N/A 20516 20531TAACCAGATTGAGTCT 66 1917 959943 N/A N/A 20615 20630 GTCTCTAATTTTACGA 531918 959953 N/A N/A 20664 20679 ACAGCTTAGAAATTGC 89 1919 959963 N/A N/A20843 20858 CTGTATTAGCTCAATA 67 1920 959973 N/A N/A 20938 20953TCTTTGTAGCAGACAG 84 1921 959983 N/A N/A 21004 21019 TTATTTTAACAGCTCA 921922 959993 N/A N/A 21410 21425 CTGCAATTCTAGACAT 29 1923 960003 N/A N/A21535 21550 CATTTCAGAGTATAAG 46 1924 960013 N/A N/A 22708 22723GATGTGAGTGAAATAA 69 1925 960023 N/A N/A 22769 22784 AAGGACATGACAGACT 681926 960033 N/A N/A 24043 24058 CCACCATCAATGCTGC 58 1927

TABLE 31Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC  99  702 959284  695  710 11913 11928 TTTGGCATCAATGAAG 69 1928 959294  817  832 12035 12050 TAGAGGTTCCCTGTGC  83 1929 9593141091 1106 16211 16226 GGGCAGGATGCTGAGA  22 1930 959324 1606 1621 2536925384 ACTCACAGACTCTTCT  68 1931 959334 1647 1662 25410 25425AGCACCTCTGAAAGAA  67 1932 959344 1678 1693 25441 25456 CGGAGGTAGCTGCACA 85 1933 959354 1926 1941 25689 25704 ATTCTAAGAACCTCAT  51 1934 959384N/A N/A  4303  4318 AAACAAACCCTCCGTC  10 1935 959394 N/A N/A  4734  4749AAATGATCATGTGGCG  94 1936 959404 N/A N/A  4768  4783 CTGACTTTTATTGTTG 74 1937 959414 N/A N/A  4871  4886 GTGAGTGTACTTTAGG  98 1938 959424 N/AN/A  5393  5408 AATGCTATCAGGTGCA  31 1939 959434 N/A N/A  5579  5594TGCACAATGACATCAT  88 1940 959444 N/A N/A  5621  5636 CTACCTGTGTCTTTTA 90 1941 959454 N/A N/A  5647  5662 ATATATTGGGCTCAAT  81 1942 959474 N/AN/A  5867  5882 ACTTTTGGCAAGGCCA  17 1943 959484 N/A N/A  7211  7226CCGCAAACAAGGTTAA  22 1944 959684 N/A N/A 12283 12298 TTTAGGTCTGGGTATA 93 1945 959694 N/A N/A 12325 12340 CCCCCTGACTATATAA  18 1946 959704 N/AN/A 12693 12708 TCTTGACCGTGTTTCC  97 1947 959734 N/A N/A 12834 12849GCATTGCATAGCCTTC  96 1948 959744 N/A N/A 12886 12901 AACCAATCCTGTTAGA 59 1949 959754 N/A N/A 12909 12924 TCTGCTTATAAAGCAC 100 1950 959774 N/AN/A 13373 13388 CTTTGCAGGCACCCCA  72 1951 959784 N/A N/A 13398 13413CTTGAATGTCACCCTT  92 1952 959804 N/A N/A 13715 13730 TTACCAAGACCGCTAG 47 1953 959824 N/A N/A 14228 14243 ACTTTTAGTATTAAAG   0 1954 959844 N/AN/A 14555 14570 AAAGTCTTAATGTGGA  88 1955 959854 N/A N/A 14670 14685CTTAGTGTCCCCATCC  76 1956 959874 N/A N/A 15767 15782 GTTAGTGTTGGTTTAT 95 1957 959884 N/A N/A 17199 17214 GTTTGACTTAGTCCGT  96 1958 959914 N/AN/A 18398 18413 AATATGTTTGGAAGTC  87 1959 959934 N/A N/A 20518 20533AGTAACCAGATTGAGT  96 1960 959954 N/A N/A 20665 20680 CACAGCTTAGAAATTG 88 1961 959964 N/A N/A 20844 20859 CCTGTATTAGCTCAAT  92 1962 959974 N/AN/A 20940 20955 CCTCTTTGTAGCAGAC  84 1963 959984 N/A N/A 21006 21021GGTTATTTTAACAGCT  85 1964 959994 N/A N/A 21412 21427 ACCTGCAATTCTAGAC 61 1965 960014 N/A N/A 22710 22725 AAGATGTGAGTGAAAT  70 1966 960024 N/AN/A 22770 22785 AAAGGACATGACAGAC  87 1967

TABLE 32Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 959275  440  455  6014  6029GAGGAACTTGCTTAAG 81 1968 959285  696  711 11914 11929 TTTTGGCATCAATGAA62 1969 959305 1066 1081 16186 16201 TCTAGCAGCTCATCTC 64 1970 9593351649 1664 25412 25427 TTAGCACCTCTGAAAG 72 1971 959345 1804 1819 2556725582 ATGTATTAGAGTTAAG 77 1972 959355 1927 1942 25690 25705CATTCTAAGAACCTCA 71 1973 959365 2183 2198 25946 25961 TTAGTTAGGTGAAAAA81 1974 959375 2261 2276 26024 26039 CACTGATTCACATAAT 70 1975 959395 N/AN/A  4737  4752 TGCAAATGATCATGTG 66 1976 959405 N/A N/A  4769  4784GCTGACTTTTATTGTT 84 1977 959415 N/A N/A  4872  4887 AGTGAGTGTACTTTAG 941978 959425 N/A N/A  5395  5410 TTAATGCTATCAGGTG 81 1979 959435 N/A N/A 5580  5595 ATGCACAATGACATCA 86 1980 959445 N/A N/A  5624  5639ATTCTACCTGTGTCTT 97 1981 959455 N/A N/A  5651  5666 TTGGATATATTGGGCT 971982 959475 N/A N/A  5868  5883 TACTTTTGGCAAGGCC 70 1983 959485 N/A N/A 7697  7712 GCACAGAGTAGGTTAA 72 1984 959655 N/A N/A 12146 12161TGGTAGCTCCTGGCAA 55 1985 959675 N/A N/A 12201 12216 TTTCCGTTAACCATCA 941986 959695 N/A N/A 12667 12682 CATCTTAGTGGCTGGG 93 1987 959705 N/A N/A12695 12710 GTTCTTGACCGTGTTT 97 1988 959715 N/A N/A 12717 12732GGTTAAACTACCGAAC 11 1989 959725 N/A N/A 12783 12798 CATGGTCTGCAAATTT 891990 959745 N/A N/A 12887 12902 AAACCAATCCTGTTAG 46 1991 959755 N/A N/A12910 12925 ATCTGCTTATAAAGCA 42 1992 959775 N/A N/A 13374 13389ACTTTGCAGGCACCCC 87 1993 959785 N/A N/A 13399 13414 GCTTGAATGTCACCCT 941994 959805 N/A N/A 13716 13731 TTTACCAAGACCGCTA 86 1995 959825 N/A N/A14230 14245 CAACTTTTAGTATTAA  0 1996 959835 N/A N/A 14417 14432TCGACACAGCATCACC 62 1997 959855 N/A N/A 14671 14686 TCTTAGTGTCCCCATC 781998 959865 N/A N/A 15209 15224 TTAGGAATATTGCCAG 93 1999 959875 N/A N/A15769 15784 GGGTTAGTGTTGGTTT 94 2000 959895 N/A N/A 17288 17303TAGGGCACCTCAAGAA  0 2001 959915 N/A N/A 18400 18415 CAAATATGTTTGGAAG 502002 959925 N/A N/A 20293 20308 CCCAATCAGAGGAAGC 41 2003 959935 N/A N/A20599 20614 TCATCATTATTACCTG 91 2004 959955 N/A N/A 20803 20818TTCAGACCAGGGTAAT 91 2005 959965 N/A N/A 20845 20860 GCCTGTATTAGCTCAA 902006 959975 N/A N/A 20941 20956 GCCTCTTTGTAGCAGA  0 2007 959995 N/A N/A21434 21449 TACTGAGAGGAAATGA 64 2008 960015 N/A N/A 22714 22729TGTAAAGATGTGAGTG 78 2009 960025 N/A N/A 22772 22787 TCAAAGGACATGACAG 802010 960037 N/A N/A 22590 22605 GAGCAACGAGGAAGGA 68 2011

TABLE 33Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959276  447  462  6021  6036 CCTGTCGGAGGAACTT37 2012 959286  699  714 11917 11932 TTGTTTTGGCATCAAT 73 2013 959296 895  910 13631 13646 AATGCATCCAAATATC  0 2014 959306 1069 1084 1618916204 TGGTCTAGCAGCTCAT 25 2015 959316 1221 1236 19061 19076TTACATAAGACATTAT 35 2016 959326 1614 1629 25377 25392 CTCAAGTGACTCACAG85 2017 959336 1650 1665 25413 25428 TTTAGCACCTCTGAAA 24 2018 9593461834 1849 25597 25612 TTTCCCAACCAGCTGA 60 2019 959356 2096 2111 2585925874 TCATCTTTGCAGACCA 90 2020 959366 2184 2199 25947 25962TTTAGTTAGGTGAAAA 57 2021 959376 2262 2277 26025 26040 TCACTGATTCACATAA86 2022 959386 N/A N/A  4306  4321 GAGAAACAAACCCTCC  0 2023 959396 N/AN/A  4738  4753 GTGCAAATGATCATGT 69 2024 959406 N/A N/A  4771  4786AAGCTGACTTTTATTG 57 2025 959416 N/A N/A  5276  5291 TCTTGGGATGCACAGG 492026 959426 N/A N/A  5397  5412 CCTTAATGCTATCAGG  0 2027 959436 N/A N/A 5581  5596 AATGCACAATGACATC 69 2028 959446 N/A N/A  5625  5640AATTCTACCTGTGTCT 82 2029 959456 N/A N/A  5652  5667 TTTGGATATATTGGGC 952030 959466 N/A N/A  5802  5817 CTACTATGGGAGCCAC 64 2031 959476 N/A N/A 5871  5886 TAATACTTTTGGCAAG 29 2032 959486 N/A N/A  7784  7799TTATAGGCGAGAGCAC  0 2033 959656 N/A N/A 12147 12162 CTGGTAGCTCCTGGCA 442034 959666 N/A N/A 12164 12179 GATTGTGCAGACAGTA 95 2035 959676 N/A N/A12202 12217 ATTTCCGTTAACCATC 93 2036 959686 N/A N/A 12288 12303TGAGTTTTAGGTCTGG 96 2037 959696 N/A N/A 12669 12684 ATCATCTTAGTGGCTG 912038 959706 N/A N/A 12696 12711 TGTTCTTGACCGTGTT 98 2039 959716 N/A N/A12719 12734 AAGGTTAAACTACCGA  6 2040 959726 N/A N/A 12785 12800TACATGGTCTGCAAAT 90 2041 959736 N/A N/A 12838 12853 CATTGCATTGCATAGC 962042 959746 N/A N/A 12888 12903 AAAACCAATCCTGTTA 47 2043 959756 N/A N/A12911 12926 CATCTGCTTATAAAGC 81 2044 959766 N/A N/A 12967 12982AAACTTTGCAGCCTAT 93 2045 959776 N/A N/A 13376 13391 AGACTTTGCAGGCACC 902046 959786 N/A N/A 13400 13415 GGCTTGAATGTCACCC 69 2047 959796 N/A N/A13697 13712 AATGCTTGTCAAAAGG 70 2048 959806 N/A N/A 13717 13732CTTTACCAAGACCGCT 82 2049 959816 N/A N/A 13747 13762 TAAGTGTTAGAACTAA 302050 959826 N/A N/A 14232 14247 ACCAACTTTTAGTATT 79 2051 959836 N/A N/A14419 14434 CATCGACACAGCATCA 59 2052 959846 N/A N/A 14557 14572CCAAAGTCTTAATGTG 53 2053 959856 N/A N/A 14676 14691 CCATCTCTTAGTGTCC 882054 959866 N/A N/A 15210 15225 CTTAGGAATATTGCCA 87 2055 959876 N/A N/A15770 15785 AGGGTTAGTGTTGGTT 89 2056 959886 N/A N/A 17202 17217TCTGTTTGACTTAGTC 70 2057 959896 N/A N/A 17290 17305 CTTAGGGCACCTCAAG 302058 959906 N/A N/A 17735 17750 TAATCTGGTCATATGG 43 2059 959916 N/A N/A18445 18460 TGCTTACGGAGCATAG  0 2060 959926 N/A N/A 20472 20487CTCTAGACGGGAAGCT 31 2061 959936 N/A N/A 20601 20616 GATCATCATTATTACC 712062 959946 N/A N/A 20652 20667 TTGCAGTGCCCTGGCC 31 2063 959956 N/A N/A20804 20819 ATTCAGACCAGGGTAA 87 2064 959966 N/A N/A 20847 20862ATGCCTGTATTAGCTC 65 2065 959976 N/A N/A 20942 20957 AGCCTCTTTGTAGCAG  52066 959986 N/A N/A 21008 21023 GAGGTTATTTTAACAG 69 2067 959996 N/A N/A21435 21450 ATACTGAGAGGAAATG 68 2068 960006 N/A N/A 21539 21554ATGACATTTCAGAGTA 88 2069 960016 N/A N/A 22715 22730 GTGTAAAGATGTGAGT 842070 960026 N/A N/A 24033 24048 TGCTGCACTCAAAGAG  0 2071 960038 N/A N/A19377 19392 TCACAAGAGACTGGAC 31 2072

TABLE 34Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959277  481  496  6055  6070 TGGTGGACATTGGCCG 5 2073 959287  707  722 11925 11940 GGTGATGGTTGTTTTG 85 2074 959297 896  911 13632 13647 GAATGCATCCAAATAT  0 2075 959307 1070 1085 1619016205 GTGGTCTAGCAGCTCA 66 2076 959317 1223 1238 19063 19078CATTACATAAGACATT 46 2077 959327 1615 1630 25378 25393 CCTCAAGTGACTCACA83 2078 959337 1651 1666 25414 25429 CTTTAGCACCTCTGAA 71 2079 9593471835 1850 25598 25613 ATTTCCCAACCAGCTG 49 2080 959357 2099 2114 2586225877 TTATCATCTTTGCAGA 48 2081 959367 2185 2200 25948 25963TTTTAGTTAGGTGAAA 43 2082 959377 2263 2278 26026 26041 CTCACTGATTCACATA79 2083 959387 N/A N/A  4307  4322 TGAGAAACAAACCCTC  0 2084 959397 N/AN/A  4739  4754 TGTGCAAATGATCATG 82 2085 959407 N/A N/A  4859  4874TAGGCTCCTGGGACCT  0 2086 959417 N/A N/A  5277  5292 ATCTTGGGATGCACAG 892087 959427 N/A N/A  5567  5582 TCATGGCTTCCAGTGT 78 2088 959437 N/A N/A 5600  5615 TTCAATGTGGCTTCTA 96 2089 959447 N/A N/A  5627  5642TTAATTCTACCTGTGT 72 2090 959457 N/A N/A  5706  5721 TGAAATATCTCATTAG 772091 959467 N/A N/A  5805  5820 TGTCTACTATGGGAGC 83 2092 959477 N/A N/A 5875  5890 ATGGTAATACTTTTGG 75 2093 959657 N/A N/A 12148 12163ACTGGTAGCTCCTGGC 78 2094 959667 N/A N/A 12165 12180 TGATTGTGCAGACAGT 972095 959677 N/A N/A 12203 12218 TATTTCCGTTAACCAT 91 2096 959687 N/A N/A12289 12304 CTGAGTTTTAGGTCTG 96 2097 959697 N/A N/A 12671 12686GAATCATCTTAGTGGC 94 2098 959707 N/A N/A 12697 12712 TTGTTCTTGACCGTGT 982099 959717 N/A N/A 12753 12768 GTGGTAAGGCATACTA 35 2100 959727 N/A N/A12789 12804 GGTGTACATGGTCTGC 97 2101 959737 N/A N/A 12839 12854GCATTGCATTGCATAG 92 2102 959747 N/A N/A 12890 12905 GGAAAACCAATCCTGT 692103 959757 N/A N/A 12927 12942 AGCTGTCTCCTCTACT 70 2104 959767 N/A N/A12968 12983 CAAACTTTGCAGCCTA 95 2105 959777 N/A N/A 13377 13392GAGACTTTGCAGGCAC 88 2106 959787 N/A N/A 13402 13417 TCGGCTTGAATGTCAC 672107 959797 N/A N/A 13700 13715 GTAAATGCTTGTCAAA 91 2108 959807 N/A N/A13720 13735 AGTCTTTACCAAGACC  0 2109 959817 N/A N/A 13911 13926CATGACATCCCAGTTC 29 2110 959827 N/A N/A 14233 14248 AACCAACTTTTAGTAT 272111 959837 N/A N/A 14420 14435 CCATCGACACAGCATC 89 2112 959847 N/A N/A14567 14582 CTACTTATCCCCAAAG 16 2113 959857 N/A N/A 14677 14692GCCATCTCTTAGTGTC 36 2114 959867 N/A N/A 15211 15226 CCTTAGGAATATTGCC 752115 959877 N/A N/A 15771 15786 GAGGGTTAGTGTTGGT 93 2116 959887 N/A N/A17218 17233 CTGGTTTGTGGGTTCT 75 2117 959897 N/A N/A 17291 17306TCTTAGGGCACCTCAA 53 2118 959907 N/A N/A 17736 17751 TTAATCTGGTCATATG  02119 959917 N/A N/A 18852 18867 ACAAAAGCGACAAGGT 33 2120 959927 N/A N/A20508 20523 TTGAGTCTCCTGACCA 65 2121 959937 N/A N/A 20602 20617CGATCATCATTATTAC 87 2122 959947 N/A N/A 20653 20668 ATTGCAGTGCCCTGGC 692123 959957 N/A N/A 20805 20820 TATTCAGACCAGGGTA 89 2124 959967 N/A N/A20848 20863 GATGCCTGTATTAGCT 72 2125 959977 N/A N/A 20944 20959GCAGCCTCTTTGTAGC  0 2126 959987 N/A N/A 21010 21025 CTGAGGTTATTTTAAC 402127 959997 N/A N/A 21436 21451 TATACTGAGAGGAAAT 47 2128 960007 N/A N/A21541 21556 ATATGACATTTCAGAG 91 2129 960017 N/A N/A 22716 22731CGTGTAAAGATGTGAG 87 2130 960027 N/A N/A 24035 24050 AATGCTGCACTCAAAG 312131 960039 N/A N/A 20215 20230 TAACAAACTATGCCTA 44 2132

TABLE 35Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959274  439  454  6013  6028 AGGAACTTGCTTAAGT73 2133 959284  695  710 11913 11928 TTTGGCATCAATGAAG 60 1928 959294 817  832 12035 12050 TAGAGGTTCCCTGTGC 77 1929 959304 1063 1078 1618316198 AGCAGCTCATCTCCCT 62 2134 959314 1091 1106 16211 16226GGGCAGGATGCTGAGA 13 1930 959324 1606 1621 25369 25384 ACTCACAGACTCTTCT72 1931 959334 1647 1662 25410 25425 AGCACCTCTGAAAGAA 65 1932 9593441678 1693 25441 25456 CGGAGGTAGCTGCACA 86 1933 959354 1926 1941 2568925704 ATTCTAAGAACCTCAT 54 1934 959364 2181 2196 25944 25959AGTTAGGTGAAAAAGG 92 2135 959374 2260 2275 26023 26038 ACTGATTCACATAATA78 2136 959384 N/A N/A  4303  4318 AAACAAACCCTCCGTC  2 1935 959394 N/AN/A  4734  4749 AAATGATCATGTGGCG 94 1936 959404 N/A N/A  4768  4783CTGACTTTTATTGTTG 72 1937 959414 N/A N/A  4871  4886 GTGAGTGTACTTTAGG 971938 959424 N/A N/A  5393  5408 AATGCTATCAGGTGCA 31 1939 959434 N/A N/A 5579  5594 TGCACAATGACATCAT 87 1940 959444 N/A N/A  5621  5636CTACCTGTGTCTTTTA 90 1941 959454 N/A N/A  5647  5662 ATATATTGGGCTCAAT 801942 959464 N/A N/A  5799  5814 CTATGGGAGCCACATG 24 2137 959474 N/A N/A 5867  5882 ACTTTTGGCAAGGCCA 23 1943 959484 N/A N/A  7211  7226CCGCAAACAAGGTTAA  0 1944 959664 N/A N/A 12157 12172 CAGACAGTAACTGGTA 962138 959674 N/A N/A 12200 12215 TTCCGTTAACCATCAA 97 2139 959684 N/A N/A12283 12298 TTTAGGTCTGGGTATA 93 1945 959694 N/A N/A 12325 12340CCCCCTGACTATATAA 26 1946 959704 N/A N/A 12693 12708 TCTTGACCGTGTTTCC 981947 959714 N/A N/A 12716 12731 GTTAAACTACCGAACG 35 2140 959724 N/A N/A12763 12778 CTATGGTAGAGTGGTA 93 2141 959734 N/A N/A 12834 12849GCATTGCATAGCCTTC 97 1948 959744 N/A N/A 12886 12901 AACCAATCCTGTTAGA 551949 959754 N/A N/A 12909 12924 TCTGCTTATAAAGCAC  0 1950 959764 N/A N/A12937 12952 GGACAATAAGAGCTGT 91 2142 959774 N/A N/A 13373 13388CTTTGCAGGCACCCCA 72 1951 959784 N/A N/A 13398 13413 CTTGAATGTCACCCTT 921952 959794 N/A N/A 13532 13547 AGCATCATTGGAAGAC 92 2143 959804 N/A N/A13715 13730 TTACCAAGACCGCTAG 57 1953 959814 N/A N/A 13744 13759GTGTTAGAACTAAGGC 94 2144 959824 N/A N/A 14228 14243 ACTTTTAGTATTAAAG  01954 959834 N/A N/A 14306 14321 TTTCTGAGCAGATAAA 66 2145 959844 N/A N/A14555 14570 AAAGTCTTAATGTGGA 87 1955 959854 N/A N/A 14670 14685CTTAGTGTCCCCATCC 77 1956 959864 N/A N/A 15208 15223 TAGGAATATTGCCAGG 892146 959874 N/A N/A 15767 15782 GTTAGTGTTGGTTTAT 94 1957 959884 N/A N/A17199 17214 GTTTGACTTAGTCCGT 95 1958 959894 N/A N/A 17228 17243AACTCTGTAGCTGGTT 41 2147 959904 N/A N/A 17603 17618 TCTTGATAGTGAATGT 732148 959914 N/A N/A 18398 18413 AATATGTTTGGAAGTC 89 1959 959924 N/A N/A20292 20307 CCAATCAGAGGAAGCC 58 2149 959934 N/A N/A 20518 20533AGTAACCAGATTGAGT 81 1960 959944 N/A N/A 20617 20632 CTGTCTCTAATTTTAC 752150 959954 N/A N/A 20665 20680 CACAGCTTAGAAATTG 87 1961 959964 N/A N/A20844 20859 CCTGTATTAGCTCAAT 92 1962 959974 N/A N/A 20940 20955CCTCTTTGTAGCAGAC 83 1963 959984 N/A N/A 21006 21021 GGTTATTTTAACAGCT 851964 959994 N/A N/A 21412 21427 ACCTGCAATTCTAGAC 54 1965 960004 N/A N/A21537 21552 GACATTTCAGAGTATA 93 2151 960014 N/A N/A 22710 22725AAGATGTGAGTGAAAT 69 1966 960024 N/A N/A 22770 22785 AAAGGACATGACAGAC 751967 960034 N/A N/A 24613 24628 GCAAATCGGATCTTTG 32 2152

TABLE 36Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 PNPLA3 SEQ CompoundStart Stop Start Stop % ID Number Site Site Site SiteSequence (5′ to 3′) Inhibition NO 915609  705  720 11923 11938TGATGGTTGTTTTGGC 98  702 959275  440  455  6014  6029 GAGGAACTTGCTTAAG80 1968 959285  696  711 11914 11929 TTTTGGCATCAATGAA 63 1969 959295 823  838 12041 12056 AGAAGGTAGAGGTTCC 48 2153 959305 1066 1081 1618616201 TCTAGCAGCTCATCTC 66 1970 959315 1093 1108 16213 16228CAGGGCAGGATGCTGA  2 2154 959325 1608 1623 25371 25386 TGACTCACAGACTCTT60 2155 959335 1649 1664 25412 25427 TTAGCACCTCTGAAAG 54 1971 9593451804 1819 25567 25582 ATGTATTAGAGTTAAG 79 1972 959355 1927 1942 2569025705 CATTCTAAGAACCTCA 68 1973 959365 2183 2198 25946 25961TTAGTTAGGTGAAAAA 69 1974 959375 2261 2276 26024 26039 CACTGATTCACATAAT73 1975 959385 N/A N/A  4305  4320 AGAAACAAACCCTCCG 70 2156 959395 N/AN/A  4737  4752 TGCAAATGATCATGTG 69 1976 959405 N/A N/A  4769  4784GCTGACTTTTATTGTT 83 1977 959415 N/A N/A  4872  4887 AGTGAGTGTACTTTAG 941978 959425 N/A N/A  5395  5410 TTAATGCTATCAGGTG 82 1979 959435 N/A N/A 5580  5595 ATGCACAATGACATCA 84 1980 959445 N/A N/A  5624  5639ATTCTACCTGTGTCTT 95 1981 959455 N/A N/A  5651  5666 TTGGATATATTGGGCT 971982 959465 N/A N/A  5800  5815 ACTATGGGAGCCACAT 26 2157 959475 N/A N/A 5868  5883 TACTTTTGGCAAGGCC 69 1983 959485 N/A N/A  7697  7712GCACAGAGTAGGTTAA 70 1984 959655 N/A N/A 12146 12161 TGGTAGCTCCTGGCAA 501985 959665 N/A N/A 12162 12177 TTGTGCAGACAGTAAC 87 2158 959675 N/A N/A12201 12216 TTTCCGTTAACCATCA 95 1986 959685 N/A N/A 12284 12299TTTTAGGTCTGGGTAT 81 2159 959695 N/A N/A 12667 12682 CATCTTAGTGGCTGGG 911987 959705 N/A N/A 12695 12710 GTTCTTGACCGTGTTT 97 1988 959715 N/A N/A12717 12732 GGTTAAACTACCGAAC 26 1989 959725 N/A N/A 12783 12798CATGGTCTGCAAATTT 89 1990 959735 N/A N/A 12837 12852 ATTGCATTGCATAGCC 952160 959745 N/A N/A 12887 12902 AAACCAATCCTGTTAG 54 1991 959755 N/A N/A12910 12925 ATCTGCTTATAAAGCA 43 1992 959765 N/A N/A 12964 12979CTTTGCAGCCTATCCC 95 2161 959775 N/A N/A 13374 13389 ACTTTGCAGGCACCCC 861993 959785 N/A N/A 13399 13414 GCTTGAATGTCACCCT 95 1994 959795 N/A N/A13534 13549 TCAGCATCATTGGAAG 60 2162 959805 N/A N/A 13716 13731TTTACCAAGACCGCTA 82 1995 959815 N/A N/A 13745 13760 AGTGTTAGAACTAAGG 932163 959825 N/A N/A 14230 14245 CAACTTTTAGTATTAA  9 1996 959835 N/A N/A14417 14432 TCGACACAGCATCACC 59 1997 959845 N/A N/A 14556 14571CAAAGTCTTAATGTGG 84 2164 959855 N/A N/A 14671 14686 TCTTAGTGTCCCCATC 781998 959865 N/A N/A 15209 15224 TTAGGAATATTGCCAG 91 1999 959875 N/A N/A15769 15784 GGGTTAGTGTTGGTTT 93 2000 959885 N/A N/A 17200 17215TGTTTGACTTAGTCCG 96 2165 959895 N/A N/A 17288 17303 TAGGGCACCTCAAGAA  02001 959905 N/A N/A 17734 17749 AATCTGGTCATATGGT 42 2166 959915 N/A N/A18400 18415 CAAATATGTTTGGAAG 55 2002 959925 N/A N/A 20293 20308CCCAATCAGAGGAAGC 57 2003 959935 N/A N/A 20599 20614 TCATCATTATTACCTG 932004 959945 N/A N/A 20651 20666 TGCAGTGCCCTGGCCT 40 2167 959955 N/A N/A20803 20818 TTCAGACCAGGGTAAT 93 2005 959965 N/A N/A 20845 20860GCCTGTATTAGCTCAA 88 2006 959975 N/A N/A 20941 20956 GCCTCTTTGTAGCAGA  02007 959985 N/A N/A 21007 21022 AGGTTATTTTAACAGC 94 2168 959995 N/A N/A21434 21449 TACTGAGAGGAAATGA 65 2008 960005 N/A N/A 21538 21553TGACATTTCAGAGTAT 87 2169 960015 N/A N/A 22714 22729 TGTAAAGATGTGAGTG 752009 960025 N/A N/A 22772 22787 TCAAAGGACATGACAG 78 2010 960037 N/A N/A22590 22605 GAGCAACGAGGAAGGA 64 2011

Example 2: Dose-Dependent Antisense Inhibition of Human PNPLA3 in A431Cells

Gapmers from Example 1 exhibiting significant in vitro inhibition ofPNPLA3 mRNA were selected and tested at various doses in A431 cells. Theantisense oligonucleotides were tested in a series of experiments thathad similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cells were plated at a densityof 10,000 cells per well and transfected free uptake with differentconcentrations of antisense oligonucleotide, as specified in the Tablesbelow. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and PNPLA3 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS36070 was used tomeasure mRNA levels. PNPLA3 mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. Results are presented as percentinhibition of PNPLA3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PNPLA3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 37 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912712 27 67 76 74 0.2 912732 54 78 88 87 <0.1 912733 45 74 85 88 <0.1912734 33 64 80 83 0.1 912756 46 72 89 92 <0.1 912757 31 62 78 86 0.2912758 38 70 85 90 0.1 912759 66 92 97 98 <0.1 912772 46 63 79 88 0.1912795 40 64 83 84 0.1 912812 43 81 88 88 <0.1 912822 81 83 92 86 <0.1912823 67 80 91 86 <0.1 912825 58 80 86 88 <0.1 912834 37 75 81 84 0.1912841 17 62 79 69 0.3 912847 70 83 90 91 <0.1 912848 80 89 90 90 <0.1912855 48 62 77 80 0.1

TABLE 38 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 68 94 94 98 <0.1 912813 57 84 90 87 <0.1 912856 60 81 91 88 <0.1912859 48 79 81 72 <0.1 912864 60 88 90 90 <0.1 912870 67 81 91 94 <0.1912871 21 67 84 89 0.2 912872 18 73 90 92 0.2 912876 43 70 87 92 0.1912933 68 89 90 90 <0.1 912940 86 91 95 96 <0.1 912941 87 94 96 96 <0.1912952 68 85 90 91 <0.1 912953 80 90 95 93 <0.1 912964 59 78 88 91 <0.1912973 53 70 87 91 <0.1 912980 54 77 84 88 <0.1 912985 23 61 81 87 0.2912988 65 83 86 89 <0.1

TABLE 39 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 72 95 97 99 <0.1 912874 78 90 96 97 <0.1 912875 64 83 92 94 <0.1912886 49 78 85 92 <0.1 912931 68 88 94 95 <0.1 912934 57 83 90 92 <0.1912936 50 78 89 89 <0.1 912938 57 73 85 87 <0.1 912943 64 84 90 93 <0.1912954 80 92 93 94 <0.1 912970 44 73 86 90 <0.1 912986 56 78 91 92 <0.1912987 79 90 92 88 <0.1 912992 21 59 74 81 0.3 915603 50 88 96 98 <0.1915623 81 96 98 98 <0.1 915643 67 89 94 96 <0.1 916602 79 92 95 96 <0.1916642 44 83 91 93 <0.1

TABLE 40 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 73 94 98 99 <0.1 915484 67 87 93 95 <0.1 915543 34 69 87 90 0.1915604 54 78 91 95 <0.1 915763 63 80 87 87 <0.1 915904 50 83 92 94 <0.1915923 63 74 82 87 <0.1 916183 33 78 89 91 0.1 916303 58 73 84 91 <0.1916343 15 72 76 87 0.2 916563 46 74 90 95 <0.1 916582 48 74 89 91 <0.1916623 64 81 91 94 <0.1 916702 45 70 78 79 <0.1 916761 46 75 85 88 <0.1916781 55 79 86 87 <0.1 916782 62 87 91 93 <0.1 916802 66 88 94 91 <0.1916822 29 72 83 87 0.1

TABLE 41 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 72 95 98 99 <0.1 915525 51 76 88 84 <0.1 915546 39 79 90 94 0.1915605 59 84 96 96 <0.1 915606 74 94 99 98 <0.1 915625 72 82 91 95 <0.1915944 36 71 75 83 0.1 916065 36 62 78 79 0.1 916144 71 86 90 92 <0.1916163 36 67 81 74 0.1 916164 82 88 89 92 <0.1 916184 60 79 87 89 <0.1916304 46 65 80 84 0.1 916324 57 77 87 92 <0.1 916344 41 70 83 88 0.1916564 38 66 88 94 0.1 916604 67 87 95 96 <0.1 916624 43 59 79 87 0.1916803 67 84 93 92 <0.1

TABLE 42 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 70 94 98 99 <0.1 915486 35 64 82 90 0.1 915487 62 89 94 95 <0.1915626 67 83 92 94 <0.1 915786 65 84 88 88 <0.1 916145 53 66 85 87 <0.1916146 62 77 86 86 <0.1 916165 71 86 89 88 <0.1 916166 71 83 87 88 <0.1916305 57 86 90 92 <0.1 916306 86 96 98 98 <0.1 916325 59 78 83 86 <0.1916345 21 47 67 73 0.4 916545 63 88 95 94 <0.1 916546 66 85 92 95 <0.1916625 47 71 84 92 <0.1 916706 22 65 80 85 0.2 916765 67 85 92 93 <0.1916845 38 71 80 87 0.1

TABLE 43 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 97 99 100 100 <0.1 915608 66 91 97 97 <0.1 915609 71 97 99 99<0.1 915627 0 26 53 62 1.3 915768 39 69 86 91 0.1 915908 49 70 80 85<0.1 915987 47 60 75 78 0.1 916008 45 69 84 83 <0.1 916187 71 82 88 92<0.1 916247 34 72 83 84 0.1 916287 31 70 90 91 0.1 916547 79 93 97 97<0.1 916566 8 45 73 81 0.5 916586 47 67 89 91 <0.1 916587 48 81 90 94<0.1 916606 18 64 87 90 0.2 916607 72 94 94 95 <0.1 916627 18 51 82 790.3 916805 18 65 78 81 0.2

TABLE 44 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 64 92 97 99 <0.1 915610 74 94 98 99 <0.1 915789 35 72 82 85 0.1915909 52 69 82 86 <0.1 915929 13 32 60 59 1.0 915969 39 54 74 74 0.2915989 46 67 81 86 0.1 916069 24 59 75 56 0.3 916148 42 71 85 80 <0.1916168 28 54 74 68 0.3 916188 22 42 72 70 0.4 916309 30 77 91 96 0.1916348 41 57 65 73 0.1 916549 64 85 94 96 <0.1 916568 54 66 81 87 <0.1916569 60 86 92 95 <0.1 916728 22 50 68 73 0.4 916788 60 89 94 96 <0.1916848 49 75 89 95 <0.1

TABLE 45 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 81 93 98 100 <0.1 915390 9 40 67 77 0.6 915611 46 80 93 88 <0.1915630 52 69 81 82 <0.1 915910 44 64 79 80 0.1 915931 83 88 89 86 <0.1916149 73 87 89 83 <0.1 916150 51 68 77 84 <0.1 916189 60 73 77 79 <0.1916310 45 77 88 95 <0.1 916330 48 67 84 86 <0.1 916550 62 85 94 97 <0.1916570 89 96 98 98 <0.1 916629 26 53 73 86 0.3 916630 52 68 87 91 <0.1916670 43 77 78 85 <0.1 916730 61 74 82 86 <0.1 916768 35 57 67 72 0.2916789 79 92 96 96 <0.1

TABLE 46 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 76 94 96 99 <0.1 915532 31 66 82 92 0.1 915612 54 77 86 90 <0.1915732 42 63 80 84 0.1 915932 45 71 88 89 <0.1 915951 26 58 71 74 0.3915991 67 84 85 85 <0.1 915992 54 78 86 87 <0.1 916112 35 67 76 78 0.1916151 51 79 87 90 <0.1 916311 36 70 81 87 0.1 916331 56 85 93 95 <0.1916332 82 91 94 96 <0.1 916390 30 41 68 64 0.5 916552 79 93 96 97 <0.1916571 53 78 90 94 <0.1 916631 48 77 86 90 <0.1 916651 81 89 94 95 <0.1916711 37 66 85 91 0.1

TABLE 47 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 58 90 98 99 <0.1 915474 51 79 90 93 <0.1 915493 48 58 83 80 0.1915494 46 73 86 90 <0.1 915674 49 72 89 93 <0.1 915933 40 63 75 79 0.1916153 68 86 89 91 <0.1 916172 85 89 87 87 <0.1 916173 81 90 91 88 <0.1916292 64 83 92 92 <0.1 916312 60 84 91 92 <0.1 916333 75 92 96 96 <0.1916572 29 62 79 88 0.2 916592 52 74 89 90 <0.1 916593 25 67 83 93 0.2916613 46 75 89 92 <0.1 916652 65 83 91 88 <0.1 916672 73 89 93 90 <0.1916772 50 61 83 89 <0.1

TABLE 48 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 50 89 96 99 <0.1 915534 0 33 66 57 1.1 915535 51 81 92 96 <0.1915634 18 67 79 84 0.2 915635 44 72 86 91 0.1 915675 36 68 82 90 0.1915735 45 68 73 84 0.1 915936 36 67 78 83 0.1 915995 78 87 90 89 <0.1915996 83 91 93 92 <0.1 916174 80 84 86 81 <0.1 916175 55 82 86 89 <0.1916334 50 82 92 94 <0.1 916335 52 76 89 93 <0.1 916575 62 88 93 93 <0.1916753 49 69 76 74 <0.1 916774 49 72 86 91 <0.1 916794 26 64 85 85 0.2916873 16 49 72 82 0.4

TABLE 49 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 57 90 97 99 <0.1 915477 47 65 88 93 0.1 915478 47 78 90 95 <0.1915497 63 68 79 86 <0.1 915637 67 91 97 98 <0.1 916037 15 47 70 61 0.6916236 80 87 90 88 <0.1 916336 52 67 81 87 <0.1 916576 50 76 89 93 <0.1916596 55 82 93 94 <0.1 916636 42 71 87 90 0.1 916637 56 85 90 93 <0.1916715 27 38 68 68 0.5 916716 35 77 89 93 0.1 916796 14 62 84 89 0.3916814 22 44 70 79 0.4 916815 56 79 87 89 <0.1 916816 33 72 83 93 0.1916874 5 34 61 70 0.8

TABLE 50 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 56 91 97 100 <0.1 915479 38 70 89 94 0.1 915618 42 63 75 85 0.1915619 65 87 96 97 <0.1 915638 31 64 80 82 0.2 915639 33 78 88 93 0.1915778 41 50 78 87 0.2 916058 26 34 73 81 0.4 916177 38 55 83 82 0.1916238 84 91 93 93 <0.1 916298 79 87 92 94 <0.1 916318 59 71 91 94 <0.1916338 71 91 94 92 <0.1 916558 73 89 94 94 <0.1 916577 41 66 78 82 0.1916578 69 85 91 93 <0.1 916638 46 84 90 92 <0.1 916757 33 60 82 88 0.2916817 31 67 82 87 0.1

TABLE 51 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)841947 50 78 89 92 <0.1 912759 75 50 85 99 <0.1 912986 54 78 90 95 <0.1915480 61 87 94 97 <0.1 915519 47 77 86 85 <0.1 915620 46 75 88 91 <0.1915780 24 76 92 94 0.1 915920 23 65 79 82 0.2 916020 45 80 85 81 <0.1916299 59 87 92 93 <0.1 916339 88 95 97 97 <0.1 916340 83 96 97 98 <0.1916559 41 68 83 89 0.1 916579 71 86 96 96 <0.1 916580 59 90 95 96 <0.1916618 10 47 70 78 0.5 916639 38 58 80 86 0.1 916778 68 87 92 94 <0.1916818 60 77 90 91 <0.1

TABLE 52 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 62.5 250 1,000 4,000 IC₅₀ Number nM nM nM nM (μM)912759 60 0 85 99 0.3 915541 48 71 89 91 <0.1 915542 50 72 86 93 <0.1915601 8 53 84 84 0.3 915602 1 56 77 91 0.4 915621 21 54 75 80 0.3915622 0 44 73 84 0.5 915922 27 64 79 85 0.2 916042 6 57 89 88 0.3916140 43 82 90 89 <0.1 916141 72 88 93 91 <0.1 916180 33 62 69 83 0.2916181 53 80 89 92 <0.1 916341 0 78 94 94 0.3 916560 72 91 95 94 <0.1916581 38 76 91 91 0.1 916601 44 80 88 90 <0.1 916701 61 83 91 93 <0.1916780 75 91 93 94 <0.1

TABLE 53 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 52 86 96 99 <0.01 959430 42 76 90 95 <0.01 959440 39 73 92 980.02 959470 46 73 89 94 <0.01 959670 52 90 96 98 <0.01 959680 50 75 9196 <0.01 959730 83 96 98 98 <0.01 959740 50 70 90 96 <0.01 959820 40 6985 92 0.02 959830 46 69 93 97 0.02 959880 34 62 85 93 0.03 960010 48 7892 95 <0.01

TABLE 54 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 53 87 98 99 <0.01 959271 55 74 91 91 <0.01 959360 7 43 79 85 0.1959361 60 87 93 94 <0.01 959411 56 76 91 94 <0.01 959441 50 81 93 97<0.01 959460 0 29 75 90 0.2 959701 62 91 97 98 <0.01 959721 80 94 97 97<0.01 959731 25 64 82 91 0.05 959741 41 65 83 91 0.02 959750 0 26 65 870.2 959761 28 60 84 91 0.05 959781 39 58 75 87 0.04 959911 20 54 78 900.1 959921 37 61 83 91 0.03 959931 48 72 89 92 <0.01 959960 11 51 79 900.1 959961 38 64 85 92 0.03

TABLE 55 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 11 71 93 98 0.1 959412 52 77 90 94 <0.01 959413 34 82 95 97 0.02959422 15 50 80 87 0.1 959432 33 60 86 95 0.04 959662 0 53 84 92 0.1959672 54 85 95 97 <0.01 959673 18 62 88 95 0.1 959682 46 77 90 91 <0.01959702 39 71 91 96 0.02 959703 81 96 99 99 <0.01 959712 4 30 75 92 0.1959713 0 53 86 96 0.1 959722 33 80 90 94 0.02 959733 31 68 92 96 0.03959782 35 63 86 94 0.03 959872 29 64 77 89 0.04 959912 25 69 89 92 0.04959982 21 61 83 91 0.1

TABLE 56 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 2 73 93 98 0.1 959363 49 82 91 91 <0.01 959393 38 71 87 95 0.02959394 27 73 91 97 0.03 959414 69 94 98 99 <0.01 959664 51 77 95 98<0.01 959674 43 74 95 98 0.02 959683 14 71 90 96 0.05 959704 57 92 98 99<0.01 959724 0 68 90 95 0.1 959734 71 93 98 98 <0.01 959814 24 76 90 950.03 959873 38 53 83 90 0.04 959874 51 82 95 97 <0.01 959884 44 77 94 97<0.01 959913 18 50 85 92 0.1 959953 6 51 85 92 0.1 959983 22 54 81 920.1 960004 10 71 92 96 0.1

TABLE 57 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 12 69 93 98 0.1 959364 32 72 89 91 0.03 959415 21 70 91 96 0.04959444 6 47 82 91 0.1 959445 32 70 92 97 0.03 959455 61 87 95 97 <0.01959675 20 56 80 94 0.1 959684 8 47 83 86 0.1 959705 77 95 98 99 <0.01959735 12 67 90 95 0.1 959764 4 32 80 92 0.1 959765 1 59 88 93 0.1959784 3 35 75 90 0.1 959785 27 72 92 96 0.03 959794 0 0 53 83 0.3959864 26 61 84 91 0.05 959885 49 81 95 96 <0.01 959914 7 43 76 89 0.1959964 17 55 83 91 0.1

TABLE 58 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μM)915609 0 73 95 97 0.1 959456 66 90 97 98 <0.01 959666 29 60 89 97 0.04959676 15 44 81 93 0.1 959686 71 92 97 97 <0.01 959695 40 75 91 93 0.02959696 21 81 90 92 0.03 959706 81 95 98 98 <0.01 959725 8 55 76 84 0.1959726 0 59 88 91 0.1 959736 46 84 94 98 <0.01 959766 22 57 83 94 0.1959776 1 53 87 93 0.1 959815 31 67 89 91 0.03 959865 6 49 84 91 0.1959875 34 74 91 92 0.02 959935 22 55 84 94 0.1 959955 0 55 83 89 0.1959985 29 71 88 93 0.03

TABLE 59 Multi-dose assay of 3-10-3 cEt gapmers in A431 cells PNPLA3 %Inhibition Compound 15.6 62.5 250 1,000 IC₅₀ Number nM nM nM nM (μm)915609 37 80 96 99 0.02 959356 40 71 87 88 0.02 959417 25 58 83 92 0.1959437 65 88 94 95 <0.01 959667 37 69 90 95 0.02 959677 29 56 82 92 0.05959687 51 79 93 97 <0.01 959697 51 75 93 95 <0.01 959707 81 94 98 98<0.01 959727 71 92 96 96 <0.01 959737 45 75 89 94 <0.01 959767 47 76 9396 <0.01 959797 32 59 87 94 0.04 959856 13 35 67 80 0.1 959876 38 75 8990 0.02 959877 40 81 89 94 <0.01 959956 25 25 66 85 0.1 960006 13 40 6883 0.1 960007 24 59 88 91 0.05

Example 3: Tolerability of Modified Oligonucleotides Targeting HumanPNPLA3 in BALB/c Mice

BALB/c mice are a multipurpose mouse model frequently utilized forsafety and efficacy testing. The mice were treated with antisenseoligonucleotides selected from studies described above and evaluated forchanges in the levels of various plasma chemistry markers.

Ionis oligonucleotides selected from the studies above were conjugatedwith 3′-THA-C₆-GalNAc₃-(3R,5S)-5-(hydroxymethyl) pyrrolidin-3-olphosphate endcap (henceforth referred to as 3′-THA).

Treatment

Groups of 6- to 7-week-old male mice were injected subcutaneously oncewith 200 mg/kg of modified oligonucleotides. One group of male BALB/cmice was injected with PBS. Mice were euthanized 72-96 hours after thesingle dose and plasma was harvested for further analysis.

To evaluate the effect of modified oligonucleotides on liver function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Beckman Coulter AU480, Brea, Calif.). Modifiedoligonucleotides that caused changes in the levels of transaminasesoutside the expected range for antisense oligonucleotides were excludedin further studies. The oligonucleotides which were considered tolerablein this study and were selected for further evaluation are presented inthe Table below. ‘Parent Oligo’ indicates the Ionis oligonucleotide thathas been described in the studies above and that was conjugated with3′-THA and tested in this study.

TABLE 60 Antisense oligonucleotides in BALB/c mouse study CompoundParent oligo ID ID 975746 916339 975747 912941 975748 916306 975755916332 975760 912848 975764 916298 975766 916552 975767 916789 975768916602 975770 912874 975771 916333 975772 916780 975775 916672 975777916558 975780 916607 975783 916338 975788 912847 975790 916778 975792912870 975794 916802 975797 916637 975799 912732 975800 912733 975803912813 975804 912823 975805 912834 975806 912855 975807 912856 975808912864 975809 912871 975810 912872 975811 912875 975813 912931 975814912934 975815 912936 975816 912938 975817 912943 975820 912988 975822915486 975829 915619 975836 915780 975840 915989 975844 916151 975849916292 975850 916299 975851 916303 975852 916309 975853 916310 975854916312 975855 916318 975856 916324 975857 916331 975858 916334 975859916335 975860 916336 975861 916549 975862 916550 975864 916563 975865916564 975866 916568 975868 916571 975869 916575 975870 916580 975871916581 975873 916586 975875 916601 975878 916624 975879 916625 975880916636 975881 916638 975883 916670 975886 916711 975887 916716 975888916774 975889 916781 975890 916782 975891 916788 975893 916815 975894916816 975895 916817 975896 916818 975897 916822 975898 916845 994288959455 994289 960010 994290 959361 994291 959271

Example 4: Effect of Antisense Inhibition of PNPLA3 in Transgenic MouseModel

A PNPLA3 transgenic mouse model from wild-type C57BL/6 generated by theUniversity of California, Irvine was used. The mouse model comprises agenomic construct containing the entire PNPLA3 gene fosmid, generouslyprovided by the University of Washington. The efficacy of Ionisoligonucleotides was evaluated in this model.

Treatment

Transgenic mice were maintained on a 12-hour light/dark cycle and werefed ad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in bufferedsaline (PBS) and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

The hPNPLA3 Tg mice were divided into groups of 2 mice each. Groupsreceived subcutaneous injections of lonis oligonucleotide at a dose of2.5 mg/kg on days 1 and 8. One group of 4 mice received subcutaneousinjections of PBS on days 1 and 8. The saline-injected group served asthe control group to which oligonucleotide-treated groups were compared.

RNA Analysis

On day 10, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 andRTS36075 were both used to measure PNPLA3 mRNA levels. Results arepresented as percent change of mRNA, relative to PBS control, normalizedwith RIBOGREEN®. As presented in the Table below, treatment with Ionisantisense oligonucleotides resulted in significant reduction of PNPLA3mRNA in comparison to the PBS control. ‘0’ indicates that theoligonucleotides did not inhibit mRNA expression.

TABLE 61 Percent inhibition of PNPLA3 mRNA in the transgenic mice liverrelative to the PBS control Inhibition Inhibition (%) (%) measuredmeasured Compound with with ID RTS36070 RTS36075 975746 99 99 975747 9999 975748 98 98 975755 99 99 975760 96 97 975764 75 83 975766 99 99975767 98 98 975768 98 98 975770 97 97 975771 98 99 975772 96 96 97577590 91 975777 85 89 975780 44 63 975783 87 90 975788 0 26 975790 0 0975792 9 34 975794 44 50 975797 0 0 975799 0 0 975800 0 5 975803 68 68975804 11 38 975805 0 0 975806 0 0 975807 0 0 975808 47 58 975809 0 19975810 12 22 975811 19 32 975813 36 39 975814 48 54 975815 78 77 97581656 56 975817 84 86 975820 35 45 975822 0 0 975829 98 98 975836 85 91975840 19 44 975844 21 31 975849 88 89 975850 41 48 975851 5 18 97585224 41 975853 0 0 975854 0 0 975855 0 0 975856 45 31 975857 73 67 97585858 40 975860 92 92 975861 66 49 975862 46 36 975864 16 21 975865 0 0975866 40 41 975868 56 48 975869 30 19 975870 0 14 975871 0 0 975875 7573 975878 18 12 975879 7 0 975880 0 0 975881 54 54 975883 77 80 97588618 28 975887 49 57 975888 10 9 975889 90 91 975890 96 98 975891 97 98975893 95 95 975894 85 87 975895 89 89 975896 91 89 975898 94 95 97589796 97 975873 99 99 994288 99 99 994289 98 99 994290 98 99 994291 95 95975859 95 96

Example 5: Tolerability of Modified Oligonucleotides Targeting HumanPNPLA3 in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with Ionis antisense oligonucleotides selected from studiesdescribed above and evaluated for changes in the levels of variousplasma chemistry markers.

Ionis oligonucleotides selected from the studies above were conjugatedwith 5′-Trishexylamino-(THA)-C₆GalNAC3 endcap (henceforth referred to as5′-THA). The Ionis oligonucleotides tested are presented in the Tablebelow. ‘Unconjugated parent ION No.’ refers to the lonis oligonucleotidedescribed in the in vitro studies above with the same sequence. ‘3′-THAcounterpart ION No.’ refers to the 3′-THA conjugated oligonucleotidewith the same sequence and evaluated in the mice studies above.

TABLE 62 5′-THA oligonucleotides tested in CD1 mice tolerability studyUnconjugated 3′-THA SEQ Compound parent ION counterpart ID ID No. ION NoSequence NO 975591 916339 975746 GGATATATTGGGCTCA 1512 975592 912941975747 TTGCATTGCATAGCCT  182 975593 916306 975748 GTGTACTTTAGGCTCC  598975600 916332 975755 CACAATGACATCATGG 1020 975605 912848 975760CGTTTTTAGTAGTCAA  141 975611 916552 975766 CCTTTTATTTCCGTTA 1024 975612916789 975767 GTAATATTCAGACCAG  899 975613 916602 975768CTAGTAAATGCTTGTC  330 975615 912874 975770 ATACTTTTGGCAAGGC  217 975616916333 975771 CTTTATTCAATGTGGC 1089 975617 916780 975772AGAAATTGCAGTGCCC 1665 975674 915619 975829 GACTTTAGGGCAGATG 1400 975704916335 975859 TAATTCTACCTGTGTC 1227 975718 916586 975873AACTTTGCAGCCTATC  605 975735 916782 975890 CTTAGAAATTGCAGTG  408 975736916788 975891 TAATATTCAGACCAGG  830 975738 916815 975893CAATTCTAGACATGGC 1313 975742 916822 975897 TATGACATTTCAGAGT  410 975743916845 975898 GTAAAGATGTGAGTGA  618 994282 959455 994288TTGGATATATTGGGCT 1982 994283 960010 994289 AGACATATGACATTTC 1745 994284959361 994290 TTTTTAGTAGTCAAGG 1757 994285 959271 994291GTTGAAGGATGGATGG 1748

Treatment

Groups of four CD1 mice each were weekly injected subcutaneously with 15mg/kg of Ionis oligonucleotides for 6 weeks, with one loading dose atday 4 (total 8 doses). One group of male CD1 mice was injectedsubcutaneously for 6 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of Ionis oligonucleotides on liver and kidneyfunction, plasma levels of transaminases (ALT and AST), albumin, totalbilirubin, and creatinine were measured at week 3 using an automatedclinical chemistry analyzer (Beckman Coulter AU480, Brea, Calif.). Theresults are presented in the Table below. Ionis oligonucleotides thatcaused changes in the levels of any of the liver or kidney functionmarkers outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 63 Plasma chemistry marker levels in CD1 mice at week 3 TotalAlbumin ALT AST bilirubin Creatinine (g/dL) (IU/L) (IU/L) (mg/dL)(mg/dL) PBS 2.9 31 64 0.4 0.1 975611 2.7 640 385 0.3 0.1 994282 2.4 7683 0.3 0.1 975592 3.0 786 942 0.5 0.1 975600 2.7 334 431 0.3 0.1 9755912.6 62 115 0.4 0.1 975718 2.4 1717 2183 1.2 0.1 994284 2.7 41 97 0.3 0.1994283 2.8 216 154 0.3 0.1 975616 3.0 69 137 0.3 0.1 975612 2.7 47 2180.4 0.1 975674 2.9 134 114 0.4 0.1 975613 2.8 60 277 0.3 0.1 975593 2.7429 405 0.4 0.1 975736 2.9 46 63 0.2 0.2 975735 2.5 46 79 0.2 0.1 9757422.6 152 96 0.2 0.1 975615 2.9 207 189 0.4 0.1 975617 2.9 65 70 0.3 0.1975605 2.9 67 92 0.3 0.1 975704 2.4 33 61 0.2 0.1 975738 2.6 43 67 0.20.1 975743 2.9 119 126 0.4 0.1 994285 2.8 400 353 0.2 0.1

Hematology Assays

Blood obtained from selected mouse groups at week 6 were sent to IDEXXBioResearch for measurement of platelet count. The results are presentedin the tables below. Ionis oligonucleotides that caused changes in theplatelet count outside the expected range for antisense oligonucleotideswere excluded in further studies.

TABLE 64 Platelet count in CD1 mice Platelet (×10³/μL) PBS 1067 9756051202 975612 1200 975613 1417 975616 1178 975617 922 975674 618 975591941 975743 1127 994282 1384 994284 1255 975704 939 975735 1039 9757361116 975738 1126 975742 808

Example 6: Tolerability of Modified Oligonucleotides Targeting HumanPNPLA3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with Ionis antisenseoligonucleotides from the studies described in the Examples above andevaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycleand fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4Sprague-Dawley rats each were weekly injected subcutaneously with 15mg/kg of Ionis oligonucleotide for 6 weeks, with one loading dose on day4 (total 8 doses). Forty eight hours after the last dose, rats wereeuthanized and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of Ionis oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Beckman Coulter AU480, Brea, Calif.). Plasma levelsof ALT (alanine transaminase) and AST (aspartate transaminase) weremeasured and the results are presented in the Table below expressed inIU/L. Plasma levels of bilirubin, creatinine, albumin, and BUN were alsomeasured using the same clinical chemistry analyzer and the results arealso presented in the Table below expressed in mg/dL. Ionisoligonucleotides that caused changes in the levels of any markers ofliver function outside the expected range for antisense oligonucleotideswere excluded in further studies.

TABLE 65 Plasma chemistry markers in Sprague-Dawley rats Total AlbuminALT AST bilirubin Creatinine BUN (g/dL) (IU/L) (IU/L) (mg/dL) (mg/dL)(mg/dL) PBS 3 35 81 0.2 0.2 12 975591 3 57 161 0.2 0.3 14 975605 4 62176 0.3 0.2 14 975612 3 106 153 0.2 0.3 13 975613 3 32 94 0.2 0.2 12975616 4 31 106 0.2 0.3 13 975617 3 49 263 0.2 0.2 12 975735 3 44 1280.2 0.2 14 975736 3 73 293 0.3 0.3 14 994282 3 41 135 0.1 0.3 12 9942843 32 95 0.1 0.2 13

Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function,urinary levels of protein and creatinine were measured using anautomated clinical chemistry analyzer (Beckman Coulter AU480, Brea,Calif.). The ratios of total protein to creatinine are presented in theTable below. Ionis oligonucleotides that caused changes in the levels ofthe ratio outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 66 Total protein to creatinine ratio in Sprague-Dawley rats PBS1.5 975591 2.0 975605 1.6 975612 1.9 975613 2.3 975616 2.0 975617 1.4975735 2.2 975736 1.1 994282 2.1 994284 2.1

Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of thestudy, and are presented in the Table below. Ionis oligonucleotides thatcaused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 67 Organ weights (g) Liver Kidney Spleen Saline 16 3 1 975591 16 41 975605 21 3 1 975612 12 3 1 975613 16 3 1 975616 15 3 1 975617 19 4 2975735 14 4 1 975736 15 3 1 994282 14 3 1 994284 15 3 1

Example 7: Effect of Antisense Inhibition of PNPLA3 in Transgenic MouseModel

Ionis oligonucleotides were tested in a multi-dose assay in the hPNPLA3Tg model.

Treatment

Transgenic mice were maintained on a 12-hour light/dark cycle and werefed ad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in bufferedsaline (PBS) and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

Study 1

The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groupsreceived subcutaneous injections of Ionis oligonucleotide at a weeklydose of 5 mg/kg, 1 mg/kg, or 0.25 mg/kg administered on days 1, 5, 8,15, and 23. One group of 4 mice received subcutaneous injections of PBSon days 1, 5, 8, 15, and 23. The saline-injected group served as thecontrol group to which oligonucleotide-treated groups were compared.

RNA Analysis

On day 26, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 andRTS36075 were both used to measure PNPLA3 mRNA levels. Results arepresented as percent change of mRNA, relative to PBS control, normalizedwith RIBOGREEN®. As presented in the Table below, treatment with Ionisantisense oligonucleotides resulted in significant dose-dependentreduction of PNPLA3 mRNA in comparison to the PBS control.

TABLE 68 Percent inhibition of PNPLA3 mRNA in the transgenic mice liverrelative to the PBS control Inhibition Inhibition measured measured byby EC₅₀ mg/kg/wk RTS36070 RTS36075 (μg/g) 975605 5 93 90 2.2 1 66 570.25 45 46 975612 5 98 99 3.1 1 89 88 0.25 34 44 975613 5 98 97 1.0 1 8785 0.25 58 56 975616 5 93 93 0.5 1 85 87 0.25 60 63 975617 5 97 97 0.3 176 78 0.25 55 53 975735 5 97 98 1.5 1 74 75 0.25 29 33 975736 5 98 980.9 1 73 71 0.25 44 45 994282 5 98 98 0.2 1 91 80 0.25 62 58 994284 5 99100 0.3 1 89 88 0.25 53 47

Study 2

The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groupsreceived subcutaneous injections of Ionis oligonucleotide at a weeklydose of 5 mg/kg, 2.5 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.25 mg/kgadministered on days 1, 5, 8, 15, and 23. One group of 4 mice receivedsubcutaneous injections of PBS on days 1, 5, 8, 15, and 23. Thesaline-injected group served as the control group to whicholigonucleotide-treated groups were compared.

RNA Analysis

On day 26, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 andRTS36075 were both used to measure PNPLA3 mRNA levels. Results arepresented as percent change of mRNA, relative to PBS control, normalizedwith RIBOGREEN®. As presented in the Table below, treatment with Ionisantisense oligonucleotides resulted in significant dose-dependentreduction of PNPLA3 mRNA in comparison to the PBS control.

TABLE 69 Percent inhibition of PNPLA3 mRNA in the transgenic mice liverrelative to the PBS control Inhibition Inhibition measured measured byby EC₅₀ EC₉₀ mg/kg/wk RTS36070 RTS36075 (μg/g) (μg/g) 975612 5 96 97 1.08.6 2.5 98 98 1 95 96 0.5 82 83 0.25 43 44 975613 5 99 99 0.9 7.7 2.5 9999 1 91 91 0.5 82 83 0.25 69 74 975616 5 96 96 1.0 9.4 2.5 94 93 1 89 890.5 81 81 0.25 73 60

Example 8: Effect of Modified Oligonucleotides Targeting Human PNPLA3 inCynomolgus Monkeys

Cynomolgus monkeys were treated with Ionis antisense oligonucleotidesselected from studies described in the Examples above. Antisenseoligonucleotide tolerability was evaluated.

Treatment

Prior to the study, the monkeys were kept in quarantine during which theanimals were observed daily for general health. The monkeys were 2-4years old and weighed 2-4 kg. Nine groups of 5 randomly assigned malecynomolgus monkeys each were injected subcutaneously with Ionisoligonucleotide or PBS in a clock-wise rotation between four differentsites on the back. The monkeys were dosed twice per week (days 1, 5, 9,and 14) for the first 2 weeks, and then subsequently once a week for 10weeks with 10 mg/kg of Ionis oligonucleotide on days 21, 28, 35, 42, 49,56, 63, 70, 77, and 84. A control group of 5 cynomolgus monkeys wasinjected with PBS in a similar manner and served as the control group.

During the study period, the monkeys were observed twice daily for signsof illness or distress. Any animal experiencing more than momentary orslight pain or distress due to the treatment, injury or illness wastreated by the veterinary staff with approved analgesics or agents torelieve the pain after consultation with the Study Director. Any animalin poor health or in a possible moribund condition was identified forfurther monitoring and possible euthanasia. Scheduled euthanasia of theanimals was conducted on day 86 approximately 48 hours after the lastdose by exsanguination while under deep anesthesia. The protocolsdescribed in the Example were approved by the Institutional Animal Careand Use Committee (IACUC).

Body and Organ Weight Measurements

To evaluate the effect of Ionis oligonucleotides on the overall healthof the animals, body and organ weights were measured. Body weights andorgan weights were measured on day 86 and the data is presented in theTable below. The results indicate that effect of treatment withantisense oligonucleotides on body and organ weights was within theexpected range for antisense oligonucleotides. Specifically, treatmentwith ION 945616 was well tolerated in terms of the body and organweights of the monkeys.

TABLE 70 Final body and organ weights in cynomolgus monkey Body Liverwith Wt Spleen Kidney gallbladder (kg) (g) (g) (g) PBS Control 2797 2.613.1 53 994284 2789 3.3 14.7 69 975605 2685 4.1 12.2 58 975616 2868 3.112.9 63 994282 2782 4.4 12.1 62 975613 2704 3.0 13.5 60 975617 2761 3.814.1 61 975735 2765 4.1 15.5 67 975736 2844 3.0 14.1 66 975612 2711 2.813.2 60

Liver Function

To evaluate the effect of lonis oligonucleotides on hepatic function,blood samples were collected from all the study groups on day 86. Themonkeys were fasted overnight prior to blood collection. Blood wascollected in tubes without anticoagulant for serum separation. The tubeswere kept at room temperature for a minimum of 90 minutes and thencentrifuged at 3000 rpm for 10 minutes to obtain serum. Levels ofvarious liver function markers were measured using a Toshiba 200FR NEOchemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and ASTwere measured and the results are presented in the Table below,expressed in IU/L. Bilirubin, a liver function marker, was similarlymeasured and is presented in the Table below, expressed in mg/dL. Theresults indicate that antisense oligonucleotides had no effect on liverfunction outside the expected range for antisense oligonucleotides.

TABLE 71 Liver function markers in cynomolgus monkey plasma ALT ASTBilirubin Albumin (IU/L) (IU/L) (mg/dL) (g/dL) PBS Control 38 55 0.2 4.3994284 64 48 0.2 3.7 975605 48 54 0.3 4.0 975616 54 57 0.3 3.9 994282 8953 0.3 4.0 975613 60 71 0.4 4.0 975617 65 61 0.3 4.0 975735 59 79 0.34.1 975736 70 56 0.3 3.9 975612 61 66 0.3 3.9

Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function,blood samples were collected from all the study groups on day 86. Themonkeys were fasted overnight prior to blood collection. Blood wascollected in tubes without anticoagulant for serum separation. The tubeswere kept at room temperature for a minimum of 90 minutes and thencentrifuged at 3000 rpm for 10 minutes to obtain serum. Levels of BUNand creatinine were measured using a Toshiba 200FR NEO chemistryanalyzer (Toshiba Co., Japan). Results are presented in the Table below,expressed in mg/dL.

The plasma chemistry data indicate that most of the Ionisoligonucleotides did not have any effect on the kidney function outsidethe expected range for antisense oligonucleotides.

TABLE 72 Plasma BUN and creatinine levels (mg/dL) in cynomolgus monkeysBUN Creatinine PBS Control 23 0.8 994284 24 0.8 975605 27 0.7 975616 210.8 994282 24 0.8 975613 23 0.9 975617 21 0.7 975735 20 0.8 975736 230.8 975612 20 0.8

Hematology

To evaluate any effect of Ionis oligonucleotides in cynomolgus monkeyson hematologic parameters, blood samples of approximately 0.5 mL ofblood was collected from each of the available study animals on day 86.The samples were collected in tubes containing K₂-EDTA. Samples wereanalyzed for red blood cell (RBC) count, white blood cells (WBC) count,individual white blood cell counts, such as that of monocytes,neutrophils, lymphocytes, as well as for platelet count, hemoglobincontent and hematocrit, using an ADVIA2120i hematology analyzer(Siemens, USA).

The data indicate the oligonucleotides did not cause any changes inhematologic parameters outside the expected range for antisenseoligonucleotides at this dose.

TABLE 73 Blood cell counts in cynomolgus monkeys RBC Platelets WBCNeutrophils Lymphocytes Monocytes (×10⁶/μL) (×10³/μL) (×10³/μL)(×10³/μL) (×10³/μL) (×10³/μL) PBS Control 6.0 342 12 3.2 7.8 0.3 9942846.0 410 10 2.7 6.7 0.3 975605 5.8 326 10 4.8 4.5 0.4 975616 6.0 362 103.4 5.8 0.3 994282 5.8 359 10 3.9 5.5 0.3 975613 5.5 327 8 2.6 5.5 0.2975617 6.1 358 10 3.1 6.4 0.3 975735 5.9 241 13 5.4 6.6 0.4 975736 5.8360 10 3.5 6.4 0.2 975612 6.2 421 11 5.1 5.7 0.2

TABLE 74 Hematologic parameters in cynomolgus monkeys Hemoglobin HCT(g/dL) (%) PBS Control 14 49 994284 14 48 975605 14 46 975616 14 49994282 14 47 975613 13 46 975617 14 49 975735 14 48 975736 14 48 97561214 49

Pro-Inflammatory Proteins Analysis

To evaluate any inflammatory effect of Ionis oligonucleotides incynomolgus monkeys, blood samples were taken for analysis. The monkeyswere fasted overnight prior to blood collection. Approximately 1.5 mL ofblood was collected from each animal and put into tubes withoutanticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min at room temperature to obtain serum. C-reactive protein(CRP), which is synthesized in the liver and which serves as a marker ofinflammation, and complement C3 were measured using a Toshiba 200FR NEOchemistry analyzer (Toshiba Co., Japan).

Example 9: Measurement of Viscosity of Antisense OligonucleotidesTargeting Human PNPLA3

The viscosity of select antisense oligonucleotides from the studiesdescribed above was measured with the aim of screening out antisenseoligonucleotides which have a viscosity of more than 40 centipoise (cP).Oligonucleotides having a viscosity greater than 40 cP would have lessthan optimal viscosity.

Oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL ofwater was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part (75 μL) of the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometer was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in the Table below, where theconcentration of each antisense oligonucleotide was 200 mg/ml, andindicate that most of the antisense oligonucleotides solutions areoptimal in their viscosity under the criterion stated above.

TABLE 75 Viscosity of antisense oligonucleotides at 200 mg/mL Viscosity(cP) 994284 21 975605 19 975616 20 994282 30 975613 24 975617 22 97573515 975736 49 975612 25

Example 10: Design of Oligonucleotides at the Site of ION 975616

Additional antisense oligonucleotides were designed targeting a PNPLA3nucleic acid that overlap the target site of ION 916333, which is theunconjugated version of ION 975616, and with different chemicalmodifications and motifs.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as 3-10-3 cEt gapmers or deoxy, MOE, and cEtoligonucleotides. The 3-10-3 cEt gapmers are 16 nucleosides in length,wherein the central gap segment comprises of ten 2′-deoxynucleosides andis flanked by wing segments on the 5′ direction and the 3′ directioncomprising three nucleosides. Each nucleoside in the 5′ wing segment andeach nucleoside in the 3′ wing segment has a cEt sugar modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines. The deoxy, MOE and (S)-cEt oligonucleotides are 16nucleosides in length wherein the nucleoside have either a MOE sugarmodification, an (S)-cEt sugar modification, or a deoxy modification.The ‘Chemistry’ column describes the sugar modifications of eacholigonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’indicates deoxyribose; the number after the ‘d’ indicates the number ofdeoxyribose; and ‘e’ indicates a MOE modification. The internucleosidelinkages throughout each gapmer are phosphorothioate (P═S) linkages. Allcytosine residues throughout each gapmer are 5-methylcytosines. “Startsite” indicates the 5′-most nucleoside to which the gapmer is targetedin the human gene sequence (SEQ ID NO: 2).

TABLE 76 Modified oligonucleotides targeting human PNPLA3 Start StopCompound SEQ Site Site Sequence Number Chemistry ID NO 5599 5614TCAATGTGGCTTCTAG 995553 kkk-d10-kkk 2170 5600 5615 TTCAATGTGGCTTCTA959437 kkk-d10-kkk 2089 5601 5616 ATTCAATGTGGCTTCT 959438 kkk-d10-kkk2171 5602 5617 TATTCAATGTGGCTTC 959439 kkk-d10-kkk 2172 5603 5618TTATTCAATGTGGCTT 959440 kkk-d10-kkk 1705 5603 5618 TTATTCAATGTGGCTT995696 k-d10-kekek 1705 5603 5618 TTATTCAATGTGGCTT 995906 kk-d9-eeekk1705 5603 5618 TTATTCAATGTGGCTT 996116 kk-d9-ekeke 1705 5604 5619TTTATTCAATGTGGCT 959441 kkk-d10-kkk 1765 5604 5619 TTTATTCAATGTGGCT995697 k-d10-kekek 1765 5604 5619 TTTATTCAATGTGGCT 995907 kk-d9-eeekk1765 5604 5619 TTTATTCAATGTGGCT 996117 kk-d9-ekeke 1765 5605 5620CTTTATTCAATGTGGC 916333 kkk-d10-kkk 1089 5605 5620 CTTTATTCAATGTGGC995698 k-d10-kekek 1089 5605 5620 CTTTATTCAATGTGGC 995908 kk-d9-eeekk1089 5605 5620 CTTTATTCAATGTGGC 996118 kk-d9-ekeke 1089 5605 5620CTTTATTCAATGTGGC 996277 kek-d9-eekk 1089 5606 5621 ACTTTATTCAATGTGG916334 kkk-d10-kkk 1158 5606 5621 ACTTTATTCAATGTGG 995699 k-d10-kekek1158 5606 5621 ACTTTATTCAATGTGG 995909 kk-d9-eeekk 1158 5606 5621ACTTTATTCAATGTGG 996119 kk-d9-ekeke 1158 5607 5622 TACTTTATTCAATGTG959442 kkk-d10-kkk 1825 5608 5623 TTACTTTATTCAATGT 959443 kkk-d10-kkk1885

The oligonucleotides were tested in a series of experiments. CulturedA-431 cells at a density of 10,000 cells per well were treated usingfree uptake with modified oligonucleotides diluted to differentconcentrations. After a treatment period of approximately 48 hours,PNPLA3 mRNA levels were measured as previously described using the HumanPNPLA3 primer-probe set RTS36070. PNPLA3 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. The IC₅₀ratios of the assays are presented in the tables below, which is theratio of the IC₅₀ of a benchmark oligonucleotide to the IC₅₀ of theoligonucleotide. Hence, a bigger value of the ratio indicates that theoligonucleotide is more active than the benchmark.

TABLE XX Efficacy of modified oligonucleotides targeting human PNPLA3Start Stop Compound IC₅₀ Site Site Number Chemistry ratio 5600 5615959437 kkk-d10-kkk 1.42 5601 5616 959438 kkk-d10-kkk 0.49 5602 5617959439 kkk-d10-kkk 0.36 5603 5618 959440 kkk-d10-kkk 0.55 5603 5618995906 kk-d9-eeekk 1.42 5604 5619 959441 kkk-d10-kkk 1.66 5605 5620916333 kkk-d10-kkk 1.96 5605 5620 995908 kk-d9-eeekk 0.70 5606 5621916334 kkk-d10-kkk 0.95 5606 5621 995909 kk-d9-eeekk 1.47

1-62. (canceled)
 63. A compound comprising a modified oligonucleotide 12to 30 linked nucleosides in length having a nucleobase sequencecomprising the nucleobase sequence SEQ ID NO: 1089, or apharmaceutically acceptable salt thereof.
 64. The compound of claim 63,wherein the modified oligonucleotide is 16 to 30 linked nucleosides inlength having a nucleobase sequence comprising the nucleobase sequenceSEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.
 65. Thecompound of claim 64, wherein the modified oligonucleotide is 16 linkednucleosides having a nucleobase sequence comprising the nucleobasesequence SEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.66. The compound of claim 63, wherein the modified oligonucleotidecomprises at least one modified internucleoside linkage, and/or at leastone modified sugar, and/or at least one modified nucleobase.
 67. Thecompound of claim 66, wherein the at least one modified intemucleosidelinkage is a phosphorothioate internucleoside linkage.
 68. The compoundof claim 66, wherein the at least one modified sugar is a bicyclicsugar.
 69. The compound of claim 68, wherein the bicyclic sugarcomprises a a 4′-CH₂—O-2′ group or a 4′-CH(CH₃)—O-2′ group or a4′-(CH₂)₂—O-2′ group.
 70. The compound of claim 66, wherein the at leastone modified sugar comprises a 2′-O(CH)₂—OCH₃ group or a 2′-O—CH₃ group.71. The compound of claim 66, wherein the at least one modifiednucleobase is a 5-methylcytosine.
 72. The compound of claim 63, whereinthe modified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of three linkednucleosides; and a 3′ wing segment consisting of three linkednucleosides; wherein the gap segment is positioned between the 5′ wingsegment and the 3′ wing segment, wherein each nucleoside of each wingsegment comprises a cEt sugar; wherein each intemucleoside linkage is aphosphorothioate linkage; wherein each cytosine is a 5-methylcytosine.73. The compound of claim 63, wherein the compound further comprises aconjugate group.
 74. The compound of claim 73, wherein the conjugategroup is positioned at the 5′ end of the modified oligonucleotide and is


75. The compound of claim 63, wherein the pharmaceutically acceptablesalt is a sodium salt.
 76. The compound of claim 63, wherein thepharmaceutically acceptable salt is a potassium salt.
 77. A compositioncomprising the compound of claims 63-76 and a pharmaceuticallyacceptable carrier.
 78. A method of treating, preventing, orameliorating a disease associated with PNPLA3 in an individualcomprising administering to the individual the composition of claim 77,thereby treating, preventing, or ameliorating the disease.
 79. Themethod of claim 78, wherein the disease is liver disease, NAFLD, hepaticsteatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis,hepatocellular carcinoma, alcoholic liver disease, alcoholicsteatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditaryhemochromatosis, or primary sclerosing cholangitis.
 80. A method ofreducing or inhibiting liver damage, steatosis, liver fibrosis, liverinflammation, liver scarring or cirrhosis, liver failure, liverenlargement, elevated transaminases, or hepatic fat accumulation in anindividual, comprising administering the composition of claim 77 to theindividual, and thereby reducing or inhibiting at least one of liverdamage, steatosis, liver fibrosis, liver inflammation, liver scarring orcirrhosis, liver failure, liver enlargement, elevated transaminases, orhepatic fat accumulation in the individual.
 81. The method of claim 80,wherein the individual has, or is at risk of having, liver disease,NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), livercirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholicsteatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditaryhemochromatosis, or primary sclerosing cholangitis.